Modulation of the Immune Response

ABSTRACT

Methods for identifying compounds that modulate the generation of regulatory T cells (Treg) in vivo and in vitro, i.e., compounds that act on the transcription factors that increase or decrease expression of Foxp3.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.14/554,536, filed on Nov. 26, 2014, which is a U.S. patent applicationSer. No. 13/745,416, filed on Jan. 18, 2013, which is a continuation ofU.S. patent application Ser. No. 12/743,680, filed on Aug. 26, 2010,which is the national stage of International Application NumberPCT/US2008/083016, filed on Nov. 10, 2008, and claims the benefit ofU.S. Provisional Patent Application Serial Nos. 60/989,309, filed onNov. 20, 2007, and 61/070,410, filed on Mar. 21, 2008, the entirecontents of which are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos.AI435801, AI043458, and NS38037 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

TECHNICAL FIELD

This invention relates to methods and compositions for increasing thenumber and/or activity of regulatory T cells (Tregs) in vivo and invitro.

BACKGROUND

Regulatory T cells (Treg) control the autoreactive components of theimmune system. Consequently, Treg dysfunction is linked to severeautoimmunity, and compounds that increase Treg numbers or activity areexpected to be useful in the treatment of autoimmune disorders such asmultiple sclerosis.

Treg cells are a specialized subset of T cells involved in the controlof pathogenic autoimmunity (Sakaguchi et al., Ann. Rev. Immunol.,22:531-562, 2004. The importance of Treg for immunoregulation ishighlighted by the immune disorders that result from Treg depletion withantibodies (Sakaguchi et al., J. Immunol. 155, 1151-64 (1995)); as aresult of the thymectomy of 3 day old newborns (Sakaguchi et al., J ExpMed. 156, 1565-76 (1982)); or treatment with diphtheria toxin intransgenic mice with a Treg-restricted expression of the diphtheriatoxin receptor (Kim et al., Nat Immunol. 8, 191-7 (2007)). In addition,Treg deficiencies have been described in several autoimmune diseasessuch as multiple sclerosis (Viglietta et al., J. Exp. Med. 199, 971-9(2004)), rheumatoid arthritis (Ehrenstein et al., J Exp Med. 200, 277-85(2004)), diabetes (Brusko et al., Diabetes. 54, 1407-14 (2005); Lindleyet al., Diabetes. 54, 92-9 (2005)), and lupus (Mudd et al., Scand. J.Immunol. 64(3):211-218 (2006)).

SUMMARY

The present invention is based, at least in part, on the discovery thattranscription factors capable of modulating (e.g., increasing ordecreasing) the expression and/or activity of the Foxp3 gene provideuseful targets for therapeutic immunomodulation. Accordingly, thepresent invention provides, inter alia, compositions and methods for theprevention or treatment of diseases caused by an abnormal (e.g.,autoimmune) or absent (e.g., including insufficient) immune response.

In one aspect, the present invention features compositions including aligand that binds specifically to an aryl hydrocarbon receptor (AHR)transcription factor, linked to a biocompatible nanoparticle. The ligandcan be, e.g., a small molecule that competes for binding to the AHRcompetitively with 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) andactivates AHR-dependent signaling. In some embodiments, the ligand is2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), tryptamine (TA),2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE), or 6-formylindolo[3,2-b]carbazole (FICZ).

In some embodiments, the composition also includes an inhibitor ofdegradation of the ligand, e.g., a monoamine oxidase inhibitor such astranylcypromine. The inhibitor can be present on (i.e., linked to) thesame nanoparticles, linked to different nanoparticles (of the same ordifferent types) or free in solution. In some embodiments, the methodsand compositions described herein include the use of a ligand that bindsspecifically to an aryl hydrocarbon receptor (AHR) transcription factor,and an inhibitor of degradation thereof, e.g., tryptamine andtranylcypromine, wherein neither is linked to a nanoparticle.

In some embodiments, the composition also includes an antibody thatselectively binds to an antigen present on a T cell, a B cell, adendritic cell, or a macrophage. The antibody can be present on (i.e.,linked to) the same nanoparticles, linked to different nanoparticles (ofthe same or different types) or free in solution.

In a further aspect, the invention features methods for increasing thenumber or activity of CD4/CD25/Foxp3-expressing T regulatory (Treg)cells in a population of T cells. The methods include contacting thepopulation of cells with a sufficient amount of a composition comprisingone or more AHR ligands selected from the group consisting of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), tryptamine (TA), and2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE), wherein the ligand is linked to a biocompatible nanoparticle, andoptionally evaluating the presence and/or number ofCD4/CD25/Foxp3-expressing cells in the population. The method results inan increase in the number and/or activity of regulatory T cells (Treg).

In some embodiments, the initial population of T cells includes one orboth of naïve T cells or CD4⁺CD62 ligand⁺ T cells. The population of Tcells can be isolated, i.e., in vitro, or in a living mammalian subject,e.g., a subject who has an autoimmune disorder, e.g., multiplesclerosis. In embodiments where the T cells are in a living subject, themethods can include administering the one or more ligands orally,mucosally, or intravenously.

In some embodiments, Treg cells generated or activated using a methoddescribed herein are administered to a subject suffering from anautoimmune disorder, in an amount sufficient to improve or ameliorate asymptom of the disorder.

Also provided herein are methods for identifying candidate compoundsthat increase generation or activity of regulatory T cells (Treg). Themethods include providing a cell expressing a reporter constructcomprising a binding sequence for the Aryl Hyrocarbon Receptor (AHR) ina mammalian Foxp3 promoter sequence, wherein said binding sequence isoperably linked to a reporter gene, for example a reporter gene selectedfrom the group consisting of luciferase, green fluorescent protein, andvariants thereof; contacting the cell with a test compound; andevaluating an effect of the test compound on expression of the reportergene. A test compound that increases or decreases expression of thereporter gene is a candidate compound that modulates generation of Treg.

The methods can optionally include measuring expression of the reporterconstruct in the presence of a known AHR ligand selected from the groupconsisting of TCDD, tryptamine, and (ITE), or a compound that binds tothe AHR competitively therewith; determining whether the candidatecompound competes for binding to the AHR with the known compound; andselecting the candidate compound if it binds the AHR competitively withthe known compound.

In one aspect, the present invention provides methods of identifyingcandidate compounds that modulate the generation of regulatory T cells(Treg). These methods include providing a cell expressing a reporterconstruct containing a binding sequence for a transcription factoroperably linked to a reporter gene. Suitable binding sequences forinclusion in the reporter construct include NKX22, AHR, EGR1, EGR2,EGR3, NGFIC, and Delta EF1. The cell is then contacted with a testcompound, and the effect of the test compound on expression of thereporter gene is evaluated. A test compound that increases or decreasesexpression of the reporter gene is a candidate compound that modulatesgeneration of Treg.

In another aspect, the present invention provides methods of identifyingcandidate compounds that modulate generation of regulatory T cells(Treg). These methods include providing a living zebrafish, e.g., azebrafish embryo, e.g., 30 minutes after the egg is laid; contacting thezebrafish with a test compound, e.g., by putting the test compound inwater in which the zebrafish is living or microinjecting the compoundinto an embryo; and evaluating an effect of the test compound on Foxp3expression in the zebrafish, wherein a test compound that increases ordecreases expression of Fox-3 in the zebrafish is a candidate compoundthat modulates generation of Treg.

In a further aspect, the present invention provides compositionscomprising transcription factor ligands capable of promoting increasedexpression, activity, or both of a Foxp3 gene.

In yet another aspect, the present invention provides methods forincreasing the numbers of Treg in a population of T cells. These methodsinclude contacting the cell with one or more transcription factorligands, e.g., selected from the group consisting of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), tryptamine (TA), and2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE), wherein the method results in an increase in the number and/oractivity of regulatory T cells (Treg). In some embodiments, the methodsinclude determining levels of Foxp3 expression in the cells.

In an additional aspect, the present invention provides methods forincreasing the numbers of Treg in a patient. These methods includeadministering one or more transcription factor ligands to a patientselected for treatment, e.g., 2,3,7,8 tetrachlorodibenzo-p-dioxin(TCDD), tryptamine (TA), and/or2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE), wherein the method results in an increase in the number and/oractivity of regulatory T cells (Treg).

As used herein, “treatment” means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. As used herein, amelioration of the symptoms of aparticular disorder refers to any lessening, whether permanent ortemporary, lasting or transient of the symptoms, that can be attributedto or associated with treatment by the compositions and methods of thepresent invention.

The terms “effective amount” and “effective to treat,” as used herein,refer to an amount or a concentration of one or more of the compositionsdescribed herein utilized for a period of time (including acute orchronic administration and periodic or continuous administration) thatis effective within the context of its administration for causing anintended effect or physiological outcome.

The term “patient” is used throughout the specification to describe ananimal, human or non-human, rodent or non-rodent, to whom treatmentaccording to the methods of the present invention is provided.Veterinary and non-veterinary applications are contemplated. The termincludes, but is not limited to, mammals, e.g., humans, other primates,pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters,cows, horses, cats, dogs, sheep and goats. Typical patients includehumans, farm animals, and domestic pets such as cats and dogs.

The term gene, as used herein refers to an isolated or purified gene.The terms “isolated” or “purified,” when applied to a nucleic acidmolecule or gene, includes nucleic acid molecules that are separatedfrom other materials, including other nucleic acids, which are presentin the natural source of the nucleic acid molecule. An “isolated”nucleic acid molecule, such as an mRNA or cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

An “isolated” or “purified” polypeptide, peptide, or protein issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the protein is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. “Substantially free” means that the preparationof a selected protein has less than about 30%, (e.g., less than 20%,10%, or 5%) by dry weight, of non-selected protein or of chemicalprecursors. Such a non-selected protein is also referred to herein as“contaminating protein”. When the isolated therapeutic proteins,peptides, or polypeptides are recombinantly produced, it can besubstantially free of culture medium, i.e., culture medium representsless than about 20%, (e.g., less than about 10% or 5%) of the volume ofthe protein preparation. The invention includes isolated or purifiedpreparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dryweight.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a bar graph of proliferative response to MT or ConA ofsplenocytes from six-month old zebrafish, 14 days after immunizationwith MT or PBS in IFA. Results are presented as the mean cpm+s.d. oftriplicates.

FIG. 1B-1D are bar graphs of expression of CD3 (1B), IL-17 (1C) and IFNg(1D) in six month old zebrafish 14 or 28 days after immunization withzebrafish brain homogenate (zCNS) or PBS in CFA, as measured by realtime PCR (mean+s.d. of triplicates).

FIG. 1E is a sequence comparison of putative FoxP3 genes of zebrafish,human and mouse. The stars indicate identity, dashes were introduced foroptimal alignment. The zinc finger, leucine zipper and forkhead domainsare highlighted with a blue, green or red box, respectively.

FIG. 1F is a bar graph of zFoxp3 expression in 293T cells cotransfectedwith constructs coding for His-labeled zFoxp3 and Renilla-labeled Foxp3.The results are normalized for the total amount of luciferase beforeprecipitation (mean+s.d. of triplicates).

FIG. 1G is a radial gene tree showing the Foxp1, Foxp2, Foxp3 and Foxp4proteins in mammals and fish, where the Ciona intestinalis Foxp sequenceis the outgroup. The branch lengths are proportional to the distancebetween the sequences. Mm, Mus musculus; Hs, Homo sapiens; Dr, Daniorerio; Ga, Gasterosteus aculeatus (stickleback); Ci, Ciona intestinalis.

FIG. 2A is a pair of bar graphs of 293T cells co-transfected withreporter constructs coding for luciferase under the control of a NF-kBor NFAT responsive promoters, and p65 NF-kB (top graph) or NFAT (bottomgraph) in the presence of vectors coding for zFoxp3, Foxp3 or control(empty vector). Luciferase activity was normalized to the renillaactivity of a co-transfected control (mean+s.d. of triplicates)

FIG. 2B is a pair of Western blots of 293T cells co-transfected withHis-tagged zFoxp3, Foxp3 and NF-kB (top graph) or HA-flagged NFAT(bottom graph) and immunoprecipitated with antibodies to His antibodies.The precipitates were resolved by PAGE-SDS and detected by western blotwith antibodies to NF-kB or HA antibodies.

FIG. 2C is a set of four graphs of MACS-purified CD4⁺CD25⁻ T-cells thatwere transduced with a bicistronic retrovirus coding for GFP and zFoxp3or an empty control retrovirus, and the GFP⁺ population was analyzed forthe surface expression of (from left to right) CD25, GITR, CD152 andCD4.

FIGS. 2D(i)-(iii) and 2E(i)-(iii) are bar graphs of MACS-purifiedCD4⁺CD25⁻ T-cells transduced with a bicistronic retrovirus coding forGFP and zFoxp3, Foxp3 or an empty control retrovirus. The GFP⁺population was analyzed for its proliferation, IL-2 and IFNg secretionupon activation with plate bound antibodies to CD3 (mean cpm orpg/ml+s.d. in triplicate wells) and (e) its suppressive activity on theproliferation and IL-2 and IFNg secretion of mouse CD4⁺CD25⁻ T-cellsactivated with plate-bound antibodies to CD3 (mean cpm or pg/ml+s.d. intriplicate wells).

FIG. 3A is a bar graph of expression of zFoxP3 determined by real timePCR in zebrafish monocytes, lymphocytes and erythrocytes sorted by FACS(mean+s.d. of triplicates).

FIG. 3B is a list of the conserved AHR binding site (CABS) on zebrafish,human and mouse Foxp3 sequence (SEQ ID NOs:4-6, respectively) indicatedand highlighted in yellow.

FIG. 3C is a pair of bar graphs of FoxP3 (left) and AHR (right)expression in FACS sorted CD4⁺Foxp3:GFP⁺ and CD4⁺Foxp3:GFP⁻ T cells asmeasured by real time PCR (mean+s.d. of triplicates normalized to GAPDHexpression).

FIG. 3D is a bar graph of zFoxp3 expression 72 hours after TCDD wasadded to the water of three-day post-fertilization zebrafish embryos, asdetermined by real time PCR (mean+s.d. of triplicates normalized toGAPDH expression).

FIG. 3E is a bar graph of frequency of CD4⁺FoxP3⁺ T cells in the CD4⁺T-cell population as determined in the draining lymph nodes by FACS fromnaïve C57BL/6J mice 11 days after after administration of 1 mg/mouseTCDD or corn oil as control, and 10 days after the mice were immunized(or not) with 100 mg/mouse of MOG₃₅₋₅₅/CFA (mean+s.d. of three mice).

FIG. 3F is a bar graph of proliferation of purified CD4⁺ T cellsstimulated with plate bound antibodies to CD3 in the presence ofdifferent concentrations of TCDD for 72 hours. Cell proliferation isindicated as cpm+s.d. in triplicate wells (*** P<0.0001, one-way ANOVA,n=3).

FIG. 3G is a set of three FACS plots of CD4⁺Foxp3:GFP T cells in theCD4⁺ T-cell population from Foxp3^(gfp) knock in mice stimulated withplate bound antibodies to CD3 and CD28 for 5 days in the presence ofnormal media (control, left panel) TCDD (middle panel) or TGFb1 (rightpanel).

FIG. 3H is a bar graph of Foxp3:GFP CD4+ T-cells positive for thedonor-specific marker CD90.2, isolated and analyzed by FACS from hostmice that received FACS-purified CD4⁺Foxp3:GFP⁻ 2D2 T cells from CD90.2Foxp3^(gfp) knock in donor mice treated with 1 mg/mouse of TCDD or cornoil as control and then immunized with 100 mg/mouse MOG₃₅₋₅₅/CFA. Theresults are presented as the mean+s.d., five mice were included pergroup. * P<0.02, unpaired t-test.

FIG. 3I Sequences corresponding to non-evolutionary conservedAHR-binding sites (NCABS)-1, -2 and -3 (SEQ ID NOs:7-9, respectively).

FIG. 3J is a schematic representation of the foxp3 gene. Arrows indicatelocation of PCR primers used in ChIP assays, exons are depicted in red,with their number indicated below them.

FIG. 3K is a bar graph of activation of the transcription of Renillaluciferase-tagged foxp3 (BACFoxp3:Ren) by expression in EL-4 cells ofmouse AHR or a constitutively activated TGFβ receptor II. Renillaactivity was normalized to the luciferase activity of a co-transfectedcontrol (mean+s.d. of triplicates).

FIG. 3L is a bar graph of ChIP analysis of the interaction of AHR withNCABS and CABS in foxp3 and cyp1a1 in CD4+ T cells treated with TCDD.(c) AHR, CYP1A1 and Foxp3 expression measured by real time PCR onCD4⁺Foxp3:GFP− T cells (GFP−), CD4⁺Foxp3:GFP+ Treg (GFP+) andCD4⁺Foxp3:GFP+ Treg treated with resveratrol for 5 h (GFP++R) (mean+s.d.of triplicates normalized to GAPDH expression).

FIG. 3M is a bar graph of ChIP analysis of the interaction of AHR to theCABS and NCABS in foxp3 and cyp1a1 in thymic CD4⁺ T cells from TCDD- orcontrol-treated mice.

FIGS. 3N(i)-(iii) are bar graphs of AHR (N(i)), CYP1A1 (N(ii)) and Foxp3(N(iii)) expression measured by real time PCR on CD4⁺Foxp3:GFP− T cells(GFP−), CD4⁺Foxp3:GFP⁺ Treg (GFP⁺) and CD4⁺Foxp3:GFP⁺ Treg treated withresveratrol for 5 h (GFP⁺+R) (mean+s.d. of triplicates normalized toGAPDH expression).

FIG. 3O is a bar graph of the effect of AHR-inactivation withresveratrol on the suppressive activity of CD4⁺Foxp3:GFP⁺ Treg that wereFACS-sorted from naive Foxp3gfp mice, assayed using CD4⁺Foxp3:GFP⁻ cellsactivated with antibodies to CD3 as effector T cells in the presence ofresveratrol. Cell proliferation is indicated as cpm+s.d. in triplicatewells.

FIG. 3P is a bar graph of MOG₃₅₋₅₅-specific suppressive activity of Tregpurified from TCDD or control-treated mice, assayed using CD4⁺Foxp3:GFP⁻2D2 T cells. Cell proliferation is indicated as cpm+s.d. in triplicatewells.

FIG. 3Q is a bar graph of suppressive activity of natural Treg, or Treginduced with TGFβ1 (TGFb1) or TCDD (TCDD). Cell proliferation isindicated as cpm+s.d. in triplicate wells.

FIG. 3R is a bar graph showing the effect of AHR activation with TCDD onthe proliferation of CD4⁺Foxp3:GFP⁺ Treg and CD4⁺Foxp3:GFP− T cells thatwere FACS-sorted from naive Foxp3gfp mice. Cell proliferation isindicated as cpm+s.d. in triplicate wells.

FIG. 3S is a bar graph of the effect of AHR-activation with TCDD on thesuppressive activity of CD4⁺Foxp3:GFP⁺ Treg that were FACS-sorted fromnaive Foxp3gfp mice, assayed using CD4⁺Foxp3:GFP⁻ cells activated withantibodies to CD3 as effector T cells in the presence of resveratrol.Cell proliferation is indicated as cpm+s.d. in triplicate wells.

FIG. 4A is a line graph showing the effect on EAE of TCDD, or oil ascontrol, administered ip to C57BL/6 mice. EAE was induced 24 hours laterby immunization with MOG₃₅₋₅₅/CFA. The course of EAE in these mice isshown as the mean EAE score+s.e.m. (P<0.001, two-way ANOVA, n=6).

FIG. 4B is a line graph showing the effect on EAE of TCDD, or oil ascontrol, administered ip to C57BL/6 wild type or AHR-mt mice. EAE wasinduced 24 hours later by immunization with MOG₃₅₋₅₅/CFA. The course ofEAE in these mice is shown as the mean EAE score+s.e.m. (P<0.001,two-way ANOVA, n=10).

FIGS. 4C-D are bar graphs of the proliferative response to MOG₃₅₋₅₅ (4C)or antibodies to CD3 (4D) of lymph node cells taken from TCDD or controltreated animals 10 days after immunization with MOG₃₅₋₅₅/CFA. Cellproliferation is indicated as cpm+s.d. in triplicate wells.

FIGS. 4E(i)-(iii) are bar graphs of cytokine secretion (expressed aspg/ml) triggered by MOG₃₅₋₅₅ in lymph node cells taken from TCDD orcontrol treated animals 10 days after immunization with MOG₃₅₋₅₅/CFA.

FIGS. 4F(i)-(ii) are bar graphs showing the decreased frequency ofCD4⁺IL17⁺ and CD4⁺IFNg⁺ T cells associated to the inhibition of EAE byAHR activation with TCDD. Draining lymph node cells were isolated fromTCDD or control treated mice 10 days after immunization withMOG₃₅₋₅₅/CFA, activated with MOG₃₅₋₅₅, and stained for intracellularFoxp3, IL-17 or IFNg. Data represent the mean percentage of cytokine⁺cells within the effector CD4⁺Foxp3⁻ T cell population+s.d., five micewere included per group. * P<0.04, unpaired t-test.

FIGS. 4G-I are bar graphs showing that AHR activation by TCDD inhibitsCNS inflammation, demyelination and axonal loss. Briefly, quantificationof the cellular infiltrate, demyelination and axonal loss on the spinalcord of TCDD-treated and control mice. Spinal cords were taken on day 19after EAE induction and stained with hematoxylin & eosin, luxol fastblue or silver stain to quantify the cellular infiltrate (g),demyelination (h) and axonal loss (i), respectively. The effect ofTCDD-treatment was analyzed using Student's t-test.

FIG. 5A is a bar graph illustrating the effects on EAE of TCDD, or oilas control, administered ip to C57BL/6 mice. EAE was induced 24 hourslater by immunization with MOG₃₅₋₅₅/CFA. The frequency of CD4⁺FoxP3⁺ Tcells in the spleen CD4⁺ T-cell population was determined 21 days afterEAE induction by FACS (mean+s.d. of five mice). * P<0.02, unpairedt-test.

FIG. 5B is a bar graph illustrating the proliferative response toMOG₃₅₋₅₅ of CD4⁺CD25⁻ lymph node cells taken from TCDD or controltreated animals 10 days after immunization with MOG₃₅₋₅₅/CFA. Cellproliferation is indicated as cpm+s.d. in triplicate wells.

FIG. 5C is a line graph of EAE scores in mice treated with CD4⁺ orCD4⁺CD25⁻ T cells (5×10⁶) that were purified from TCDD or controltreated mice 10 days after immunization with MOG₃₅₋₅₅/CFA. After 1 day,EAE was induced in the recipient mice with MOG₃₅₋₅₅/CFA. The course ofEAE in these mice is shown as the mean EAE score+s.e.m. (P<0.001,two-way ANOVA, n=4).

FIG. 5D is a bar graph showing the proliferative response of lymph nodecells taken from TCDD-treated animals 10 days after immunization withMOG₃₅₋₅₅/CFA, activated in vitro with MOG₃₅₋₅₅ in the presence ofblocking antibodies to IL-4, IL-10, TGFb or isotype control. Cellproliferation is indicated as cpm+s.d. in triplicate wells.

FIG. 5E is a line graph of EAE in naïve wild type (WT) or dominantnegative TGFbRII mice injected with CD4⁺ T cells (5×10⁶) purified fromTCDD or control treated mice 10 days after immunization withMOG₃₅₋₅₅/CFA. After 1 day, EAE was induced in the recipient mice withMOG₃₅₋₅₅/CFA. The course of EAE in these mice is shown as the mean EAEscore+s.e.m. (P<0.001, two-way ANOVA, n=4).

FIG. 5F is a bar graph of proliferation to MOG₃₅₋₅₅ of CD4⁺Foxp3:GFP⁻lymph node cells from TCDD- or control-treated Foxp3gfp mice, (cpm+s.d.in triplicate wells).

FIG. 5G is a bar graph of the recall cytokine response to MOG₃₅₋₅₅ ofCD4⁺Foxp3:GFP⁻ lymph node cells taken from TCDD or control treatedFoxp3gfp mice 10 days after immunization with MOG₃₅₋₅₅/CFA. Cytokinesecretion is expressed as pg/m in triplicate wells.

FIG. 5H is a line graph of clinical EAE scores. TCDD-treated mice showeda significant delay in the onset of EAE (P=0.03, Student's t-test, n=9).

FIG. 5I is a pair of FACS plots from draining lymph node cells recoveredon day 18, stimulated with PMA/ionomycin and stained for CD4 andintracellular IL-17 and IFNγ. The numbers in the quadrants showpercentages of cytokine positive cells in the CD4⁺Foxp3:GFP⁻ T cellgate. Treatment with TCDD led to a significant decrease in the frequencyof CD4⁺ IL-17⁺ T cells (P=0.03, Student's t-test, n=4).

FIGS. 6A-C are bar graphs showing that endogenous AHR ligands controlEAE development. 6A, the frequency of CD4⁺Foxp3⁺ T cells in the CD4⁺T-cell population was determined by FACS in the blood of wild typeC57BL/6 and AHR-mt mice, and is presented as the mean+s.d. (n=5-11,P<0.03 t-test). 6B, EAE was induced in wild type C57BL/6J and AHR-mtmice by immunization with MOG₃₅₋₅₅/CFA. The course of EAE is shown asthe mean EAE score+s.e.m. (P<0.001, two-way ANOVA, n=6-8. 6C, ITE (100mg/mouse), TA (100 mg/mouse) or PBS as a control were administered ondaily basis to C57BL/6 mice. One day after the first administration, EAEwas induced by immunization with MOG₃₅₋₅₅/CFA. The course of EAE isshown as the mean EAE score+s.e.m. (P<0.001, two-way ANOVA, n=9).

FIGS. 7A-B show the results of phylogenetic footprinting for theidentification of putative TFBS. The genomic sequences of human, mouse,rat, dog and zebrafish Foxp3 were analyzed by phylogenetic footprinting.7A presents a phylogenetic tree; Tree distances are in # ofsubstitutions per 1 kb. 7B is a graph illustrating the dynamicvisualization of the location of putative TFBS conserved between human(SEQ ID NO: 1) and zebrafish (SEQ ID NO: 2).

FIGS. 8A-E and 9A-E are bar graphs showing expression levels oftranscription factors in cells transfected with Foxp3. FIGS. 8A-E showan increase in FOXP3 (8A) and NKX2.2 (8B), and a decrease in EGR1 (8C),EGR2 (8D), and EGR3 (8E) in transfected cells. FIGS. 9A-D show anincrease in NKX2.2, and a decrease in EGR1, EGR2, and EGR3 expression inFoxp3 transfected cells at days 3 and 6.

FIG. 10 is a list of the binding sites of NKX22, EGR1, EGR2, EGR3, NGFICand Delta EF1 in the mouse Foxp3 gene, relative to the numbering of thegene as shown in GenBank Acc. No. NT_039700.6. (SEQ ID NOs:10-137).

FIGS. 11A-G are a list of all the AHR binding sites on the mouse Foxp3gene, GenBank Acc. No. NT_039700.6; SEQ ID NO: 138.

FIGS. 12A-F is the genomic sequence of the zebrafish Foxp3, NW_644989.1(SEQ ID NO: 139).

FIG. 13 is a bar graph showing the effect of the monoamine oxidaseinhibitor Tranylcypromine on the suppression of EAE by TA. C57BL/6 mice(4-7/group) were treated with TA or TA and Tranylcypromine (INH), EAEwas induced and the mice were monitored for the development of EAE.

FIG. 14 is a bar graph of the expression of a renilla-tagged mouse foxp3locus on zebrafish embryos in the presence of increasing amounts ofTCDD.

FIGS. 15A-B are bar graphs of IP- and oral-ITE suppression of EAE. EAEwas induced in B6 mice (n=10), the mice were treated daily with ITE (200g/mouse) or vehicle administered orally or intraperitoneally, and themice were scored for EAE development on daily basis.

FIGS. 16A-B and 17A-B are FACS plots (16A-B) and bar graphs (17A-B)showing the induction of FoxP3⁺ T_(reg) by IP administration of ITE. EAEwas induced in B6 mice (n=10), the mice were treated with ITE orvehicle, and T_(reg) levels were analyzed by FACS on splenocytes at day17 after EAE induction.

FIGS. 18A-B and 19A-B are FACS plots (18A-B) and bar graphs (19A-B)showing the induction of FoxP3⁺ T_(reg) by oral administration of ITE.EAE was induced in B6 mice (n=10), the mice were treated with ITE orvehicle, and T_(reg) levels were analyzed by FACS on splenocytes at day17 after EAE induction.

FIGS. 20A and B are FACS plots (20A) and bar graphs (20B) showing the:induction of FoxP:GFP3⁺ T_(reg) by IP administration of ITE toFoxP3^(gfp) mice. B6 mice (n=3), the mice were treated with ITE orvehicle, immunized with CFA/MOG₃₅₋₅₅ and T_(reg) levels were analyzed byFACS on splenocytes 10 days after immunization.

FIGS. 21A and B are FACS plots (21A) and bar graphs (21B) showing the:induction of FoxP:GFP3⁺ T_(reg) by IP administration of ITE toFoxP3^(gfp) mice. B6 mice (n=3), the mice were treated with ITE orvehicle, immunized with CFA/MOG₃₅₋₅₅ and T_(reg) levels were analyzed byFACS on blood 10 days after immunization

FIGS. 22A and B are line graphs showing that IP-ITE suppresses therecall response to MOG. EAE was induced in B6 mice (n=3), the mice weretreated with ITE or vehicle, and the recall response to MOG₃₅₋₅₅ (22A)or αCD3 (22B) on splenocytes was analyzed at day 17 after EAE induction.

FIG. 22C is a set of six bar graphs of cytokine expression in the samecells as in 22A and B.

FIGS. 23A and B are a line graph (23A) and a set of six bar graphs (23B)showing that IP-ITE interferes with the generation of T_(H)1 and T_(H)17cells. EAE was induced in B6 mice (n=3), the mice were treated with ITEor vehicle, and the induction of T_(H)1 and T_(H)17 cells was followedat day 17 after EAE induction by FACS and by ELISA.

FIGS. 24A and B are FACS plots (24A) and bar graphs (24B) showing thatoral-ITE decreases the recall response to MOG. EAE was induced in B6mice (n=3), the mice were treated with ITE or vehicle, and the recallresponse to MOG₃₅₋₅₅ (left panels) or αCD3 (right panels) on splenocyteswas analyzed at day 17 after EAE induction.

FIGS. 25A and B are FACS plots IP-ITE increases the Treg:Teff ratio ofMOG₃₅₋₅₅ specific T cells. FoxP3^(gfp) mice (n=3) were immunized withMOG₃₅₋₅₅ and treated with ITE (lower panels of 25A) or vehicle (upperpanels of 25A), and the frequency of MOG₃₅₋₅₅-specific Treg and Teffcells was followed by FACS at day 10 after immunization.

FIG. 26 is a set of four line graphs showing that IP-ITE suppresses theCD4+ T cell response to MOG₃₅₋₅₅. FoxP3^(gfp) mice (n=3) were immunizedwith MOG₃₅₋₅₅ and treated with ITE or vehicle, and the recall responseof sorted T cell populations was analyzed at day 10 after immunization.

FIGS. 27A and B are bar graphs showing that IP-ITE potentiatesMOG₃₅₋₅₅-specific T_(reg) activity. FoxP3^(gfp) mice (n=3) wereimmunized with MOG₃₅₋₅₅ and treated with ITE or vehicle, FoxP3:GFP⁺T_(reg) were FACS-sorted 10 days after immunization and theirsuppressive activity was evaluated using 2D2 FoxP3:GFP− T cells asresponders. FIG. 27C is a bar graph showing that this effect could beinhibited with antibodies blocking antibodies to TGFb1.

FIG. 28 is a pair of line graphs showing that CD4⁺ T cells can transferthe protection against EAE induced by IP and oral-ITE. T cells weresorted out from EAE B6 mice treated with vehicle or ITE on day 20 afterdisease induction, 3 million cells were transferred to naïve B6 mice(n=5) and EAE was induced in the recipients 2 days after the celltransfer.

FIG. 29 is a set of six FACS plots showing modulation of APC populationsby IP-ITE. EAE was induced in B6 mice (n=3), the mice were treated withITE or vehicle, and APC were analyzed by FACS on splenocytes at day 17after EAE induction.

FIG. 30 is a line graph showing that weekly administration of ITE failsto suppress EAE development. EAE was induced in B6 mice (n=10) and themice were treated daily or weekly with 200 μg/mouse of ITE or vehicle.

FIG. 31 is a schematic diagram of gold nanoparticles for AHR-liganddelivery.

FIG. 32 is a pair of graphs showing the functionality of goldnanoparticles containing AHR-ligands. Nanoparticles were evaluated fortheir ability to activate the luciferase activity of an AHR reportercell line.

FIG. 33 is a bar graph showing modulation of EAE by AHR-ligandnanoparticles. EAE was induced in B6 mice (n=5), the mice were treatedwith nanoparticles weekly starting from day 0, and the animals werefollowed for signs of EAE.

FIG. 34 is a set of nine FACS plots showing induction of FoxP3⁺ Treg bynanoparticle-mediated delivery of ITE. EAE was induced in B6 mice (n=5),the mice were treated with nanoparticles weekly starting from day 0, andT_(reg) levels were analyzed by FACS on splenocytes at day 22 after EAEinduction.

FIGS. 35A and B show nanoparticle-mediated delivery of ITE suppressesthe recall response to MOG. EAE was induced in B6 mice (n=5), the micewere treated with ITE or vehicle, and the recall response to MOG₃₅₋₅₅(35A, top) or αCD3 (35A, bottom) on splenocytes was analyzed at day 22after EAE induction. FIG. 35B shows the cytokine response in the samecells.

FIG. 36 is a set of FACS plots showing induction of human CD4+ FoxP3+ Tcells by TCDD. CD4⁺CD62L⁺ CD45RO⁻ T cells were isolated by FACS anddifferentiated in vitro for 5 days with antibodies to CD3 and CD28 inthe presence of TCDD 100 nM or TGFβ1 2.5 ng/ml or both.

FIG. 37 is a set of four FACS plots demonstrating heterogeneity in theinduction of human CD4⁺ FoxP3⁺ T cells by TCDD. CD4⁺CD62L⁺CD45RO⁻ Tcells were isolated by FACS and differentiated in vitro for 5 days withantibodies to CD3 and CD28 in the presence of TCDD 100 nM or TGFβ1 2.5ng/ml or both.

FIG. 38 is a pair of bar graphs showing activation of human T cells inthe presence of TCDD induces suppressive T cells. CD4⁺CD62L+CD45RO⁻ Tcells were isolated by FACS and differentiated in vitro for 5 days withantibodies to CD3 and CD28 in the presence of TCDD or TGFβ1 2.5 ng/ml orboth, and after re-purification by FACS, CD4⁺CD25^(High) andCD4⁺CD25^(Low) T cells were assayed for their suppressive activity onnon-treated effector T cells activated with antibodies to CD28 and CD3.

FIG. 39 is a: FoxP3 expression by in vitro differentiated human T cells.CD4⁺CD62L⁺CD45RO⁻ T cells were isolated by FACS and differentiated invitro for 5 days with antibodies to CD3 and CD28 in the presence of TCDD100 nM or TGFβ1 2.5 ng/ml or both, and FoxP3 expression was analyzed byreal-time PCR on CD25^(High) or CD25^(Low) sorted CD4 T cells.

FIG. 40 is a pair of bar graphs showing AHR expression by in vitrodifferentiated human T cells. CD4⁺CD62L⁺CD45RO⁻ T cells were isolated byFACS and differentiated in vitro for 5 days with antibodies to CD3 andCD28 in the presence of TCDD 100 nM or TGFβ1 2.5 ng/ml or both, and AHRexpression was analyzed by real-time PCR on CD25^(High) or CD25^(Low)sorted CD4 T cells.

FIG. 41 is a pair of bar graphs showing IL-10 production by in vitrodifferentiated human T cells. CD4⁺CD62L⁺CD45RO⁻ T cells were isolated byFACS and differentiated in vitro for 5 days with antibodies to CD3 andCD28 in the presence of TCDD 100 nM or TGFβ1 2.5 ng/ml or both, andIL-10 production was analzyed by real-time PCR on CD25^(High) orCD25^(Low) sorted CD4 T cells.

FIG. 42 is a bar graph showing the suppressive activity of humanCD4⁺CD25^(High) T cells induced with TCDD is dependent on IL-10.CD4⁺CD62L⁺CD45RO⁻ T cells were isolated by FACS and differentiated invitro for 5 days with antibodies to CD3 and CD28 in the presence of TCDDor TGFβ1 2.5 ng/ml or both, and after re-purification by FACS,CD4⁺CD25^(High) T cells were assayed for their suppressive activity onnon-treated effector T cells activated with antibodies to CD28 and CD3in the presence of blocking antibodies to IL-10.

DETAILED DESCRIPTION

Because of the importance of the central role Tregs play inimmunomodulation, characterization of the pathways and identification ofcompounds capable of modulating these pathways, e.g., to promote thegeneration (e.g., differentiation of cells to or towards) Treg cells orthat promote increased activity of Tregs is important for the treatmentof, e.g., autoimmunity, infections and cancer.

The present invention provides, inter alia, compositions and methodsuseful for therapeutic immunomodulation.

Accordingly, the present invention is based, at least in part, on thediscovery that modulation of the AhR by compounds described herein canbe used to modulate (e.g., increase or decrease the number and/oractivity of) immunomodulatory cells in vitro and in vivo.

In some embodiments, the present invention is based on theidentification of the ligand-activated transcription factor arylhydrocarbon receptor (AHR) as a Foxp3 dependent regulator of Tregdifferentiation (e.g., generation) and/or activity in vitro and in vivo.Also described herein are ligands of a transcription factor (e.g., AHR)that cause increased Treg expression and/or activity. More specifically,the data presented herein demonstrates the use of AHR-specific ligands,e.g., the high affinity AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD), tryptamine (TA), and/or2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE), to promote an increase in the number and/or activity of Tregimmunomodulatory cells, which will be useful to suppress the immuneresponse in the treatment of diseases or disorders caused by an abnormal(e.g., an excessive, elevated, or inappropriate) immune response, e.g.,an autoimmune disease or disorder. Surprisingly, effective doses of TCDDcan be administered intravenously or orally.

Other potentially useful AHR transcription factor ligands are describedin Denison and Nagy, Ann. Rev. Pharmacol. Toxicol., 43:309-34, 2003, andreferences cited herein, all of which are incorporated herein in theirentirety. Other such molecules include planar, hydrophobic HAHs (such asthe polyhalogenated dibenzo-pdioxins, dibenzofurans, and biphenyls) andPAHs (such as 3-methylcholanthrene, benzo(a)pyrene, benzanthracenes, andbenzoflavones), and related compounds. (Denison and Nagy, 2003, supra).Nagy et al., Toxicol. Sci. 65:200-10 (2002), described a high-throughputscreen useful for identifying and confirming other ligands. See alsoNagy et al., Biochem. 41:861-68 (2002). In some embodiments, thoseligands useful in the present invention are those that bindcompetitively with TCDD, TA, and/or ITE.

In some embodiments, the present invention provides methods useful foridentifying transcription factors (e.g., ligand-activated transcriptionfactors) and/or ligands (e.g., ligands capable of promoting an increasedassociation between a ligand-activated transcription factor and Foxp3)capable of modulating (e.g., increasing or decreasing) Foxp3 expressionor activity.

Therapeutic Sequences

As stated above, the present invention includes the identification ofspecific transcription factor binding sites in the Foxp3 gene. Thesebinding sites include, i.e., NKX22, AHR, EGR1, EGR2, EGR3, NGFIC, andDelta EF1. As described herein, manipulating activity and/or levels ofthose TFs can alter expression of Foxp3, and thus modulate (e.g.,promote) generation and/or increased activity of Treg in vivo and invitro. Compounds that modulate the activity and/or levels of those TFsto increase generation and/or activity of Treg are useful, e.g., in thetreatment of disorders in which it is desirable to decrease an aberrantimmune response, e.g., autoimmune diseases.

Sequences useful in the methods described herein include, but are notlimited to, e.g., NKX22, AHR, EGR1, EGR2, EGR3, NGFIC and Delta EF1sequences, and TF binding sequences therefore, all of which are known inthe art. In some embodiments, the methods include the use of nucleicacids or polypeptides that are at least 80% identical to a human NKX22,AHR, EGR1, EGR2, EGR3, NGFIC, or Delta EF1 sequence, e.g., at least 80%,85%, 90%, or 95% identical to a human sequence as described herein.

To determine the percent identity of two sequences, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inone or both of a first and a second amino acid or nucleic acid sequencefor optimal alignment and non-homologous sequences can be disregardedfor comparison purposes). The length of a reference sequence aligned forcomparison purposes is at least 60%, e.g., at least 70%, 80%, 90%, 100%of the length of the reference sequence. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In the present methods, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available on the worldwide web at www.gcg.com), using a Blossum 62 scoring matrix with a gappenalty of 12, a gap extend penalty of 4, and a frameshift gap penaltyof 5.

Active fragments of TFs useful in the methods described herein are thosefragments that bind to the same DNA sequence (e.g., promoter sequence)that the full-length TF binds to, and has at least 30% of thetranscription initiating activity of the full-length TF, e.g., at least40%, 50%, 60%, 70%, 80%, 90% or more of the activity of the full-lengthprotein, on the same promoters and the same genes as the full-lengthprotein.

Foxp3

At least in some species, Treg differentiation and function is driven bythe transcription factor Foxp3 (Fontenot et al., Nat. Immunol.,4:330-336, 2003; Hori et al., Science, 299:1057-61, 2003). Foxp3 mayalso be important for human Treg; mutations in Foxp3 have been linked tovarious immunological conditions (e.g., autoimmune conditions), forexample, autoimmune syndrome immune dysregulation, polyendocrinopathy,and enteropathy X-linked (IPEX) (Chatila et al., J. Clin. Invest.,106:R75-81 (2000); Gavin et al., Proc. Natl. Acad. Sci. U.S.A., 103:6659-64 (2006)). In humans, Foxp3-negative Tregs have also beendescribed, see, e.g., Roncarolo and Gregori, Eur J Immunol. 38, 925(2008).

Exemplary human Foxp3 mRNA sequences are known in the art and includeGenbank Acc. No. NM_014009.2; the amino acid sequence of the protein isGenbank Acc. No. NP_054728.2. The sequence of the human Foxp3 gene canbe found at NC_000023.9; the mouse gene is at NT_039700.6. The Foxp3promoter has been identified and sequenced, see, e.g., Mantel et al., J.Immunol. 176 (6): 3593 (2006). All of the binding sites for AHR in themouse Foxp3 gene are highlighted, e.g., in FIGS. 11A-G; the bindingsites for the other TFs are identified in FIGS. 10A-B.

Transcription Factors that Increase Transcription of Foxp3

As described herein, NKX22, AHR, and Delta EF1 increase transcription ofFoxp3. Therefore, compounds that increase levels and/or activity ofthese TFs would increase the generation and/or activity of Treg.Conversely, compounds that decrease levels and/or activity of these TFswould be expected to decrease generation of Tregs, thereby increasingthe immune response.

AHR

Exemplary human AhR mRNA sequences are known in the art and includeGenbank Acc. No. NM_001621.3; the amino acid sequence of the protein isGenbank Acc. No. NP_001612.1. Active fragments of AhR are DNA bindingfragments with transcription activity, and contain at least one PASregion, e.g., amino acids 122-224 or 282-381 of NP_001612.1. Consensusrecognition sequences that bind AhR include the sequence TNGCGTG.

DeltaEF1

Exemplary human DeltaEF1 mRNA sequences are known in the art and includeGenbank Acc. No. NM_030751.3; the amino acid sequence of the protein isGenbank Acc. No. NP_110378.2. Consensus recognition sequences that bindDeltaEF1 include the sequences CACCT and CACCTG (Sekido et al., GenesCells 2:771-783 (1997)).

NKX2.2

Exemplary human NKX2.2 mRNA sequences are known in the art and includeGenbank Acc. No. NM_002509.2; the amino acid sequence of the protein isGenbank Acc. No. NP_002500.1. Consensus recognition sequences that bindNKX2.2 include the sequences ACTTGAT and T(T/C)AAGT(A/G)(C/G)TT (SEQ IDNO: 140)(Watada et al., Proc. Natl. Acad. Sci. U.S.A. 97(17):9443-9448(2000))

Transcription Factors that Decrease Transcription of Foxp3

As described herein, EGR1, EGR2, EGR3, and NGFIC (EGR4) decreasetranscription of Foxp3. Therefore, compounds that increase levels and/oractivity of these TFs would decrease generation of Tregs, therebyincreasing the immune response. Conversely, compounds that decreaselevels and/or activity of these TFs would be expected to increasegeneration of Tregs, reducing the immune response.

EGR1

The sequence of human egr1 protein is available in the GenBank databaseat Accession No. NP 001955.1; the mRNA is at Accession No. NM 001964.2.Additional information regarding egr1 can be found on the internet atncbi.nlm.nih.gov, in the UniGene database at UniGene Hs.326035, and inthe Entrez Gene database at GeneID: 1958. Consensus recognitionsequences that bind EGR1 include the sequence 5′GCG(G/T)GGGCG3′ (SEQ IDNO:141) (Nakagama et al., Mol. Cell. Biol., 15 (3):1489-1498 (1995)).

Active fragments of egr1 include those portions of the protein that bindDNA, e.g., one or more of the two C2H2 type DNA-binding zinc fingers(see, e.g., Sukhatme et al., 1988, supra), e.g., amino acids 338-362and/or 368-390 of GenBank Acc. No. NP_001955.1. Exemplary activefragments are described in Huang et al., Cancer Res. 1995;55(21):5054-5062, and in Jain et al., J. Biol. Chem. 1996;271(23):13530-6.

Inhibitors of egr-1 are described in WO2007/118157.

EGR2

The sequence of human egr2 protein is available in the GenBank databaseat Accession No. NP_000390.2; the mRNA is at Accession No. NM_000399.2.

Consensus recognition sequences that bind EGR2 include the sequencesGCGGGGGCG (SEQ ID NO: 142) and T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (SEQ IDNO: 143)(lowercase letters indicate bases selected less frequently)(Swirnoff and Milbrandt, Mol. Cell. Biol. 15:2275-2287 (1995)).

EGR3

The sequence of human egr3 protein is available in the GenBank databaseat Accession No. NP_004421.2; the mRNA is at Accession No. NM_004430.2.The gen

Consensus recognition sequences that bind EGR3 include the sequencesGCGGGGGCG (SEQ ID NO: 142) and T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (SEQ IDNO: 143) (lowercase letters indicate bases selected less frequently)(Swimoff and Milbrandt, Mol. Cell. Biol. 15:2275-2287 (1995)).

NGFIC (EGR4)

Exemplary human NGFIC mRNA sequences are known in the art and includeGenbank Acc. No. NM_001965.2; the amino acid sequence of the protein isGenbank Acc. No. NP_001956.2. See Crosby et al., Mol Cell Biol.11(8):3835-41 (1991).

Consensus recognition sequences that bind NGFIC include the sequencesGCGGGGGCG (SEQ ID NO: 142) and T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (SEQ IDNO: 143) (lowercase letters indicate bases selected less frequently)(Swirnoff and Milbrandt, Mol. Cell. Biol. 15:2275-2287 (1995)).

Methods of Identifying Compounds that Modulate Expression, Levels orActivity of One or More of NKX22, AHR, EGR1, EGR2, EGR3, NGFIC and DeltaEF1

A number of methods are known in the art for evaluating whether acompound alters expression, levels or activity of one or more of NKX22,AHR, EGR1, EGR2, EGR3, NGFIC, and/or Delta EF1.

Methods of assessing expression are well known in the art and include,but are not limited to, Northern analysis, ribonuclease protectionassay, reverse transcription-polymerase chain reaction (RT-PCR), realtime PCR, and RNA in situ hybridization (see, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., Cold Spring HarborLaboratory Press (2001)). Levels of peptides can be monitored by, e.g.,Western analysis, immunoassay, or in situ hybridization. Activity, e.g.,altered promoter binding and/or transcription activity, can bedetermined by, e.g., electrophoretic mobility shift assay, DNAfootprinting, reporter gene assay, or a serine, threonine, or tyrosinephosphorylation assay. In some embodiments, the effect of a testcompound on expression, level or activity is observed as a change inglucose tolerance or insulin secretion of the cell, cell extract,co-culture, explant, or subject. In some embodiments, the effect of atest compound on expression, level, or activity of one or more of NKX22,AHR, EGR1, EGR2, EGR3, NGFIC, and/or Delta EF1, is evaluated in atransgenic cell or non-human animal, or explant, tissue, or cell derivedtherefrom, having altered glucose tolerance or insulin secretion, andcan be compared to a control, e.g., wild-type animal, or explant or cellderived therefrom.

The effect of a test compound on expression, level, or activity can beevaluated in a cell, e.g., a cultured mammalian cell, a pancreatic betacell, cell lysate, or subject, e.g., a non-human experimental mammalsuch as a rodent, e.g., a rat, mouse, or rabbit, or a cell, tissue, ororgan explant, e.g., pancreas or pancreatic cells.

In some embodiments, the ability of a test compound to modulate level,expression or activity of one or more of NKX22, AHR, EGR1, EGR2, EGR3,NGFIC and/or Delta EF1 is evaluated in a knockout animal, or otheranimal having decreased expression, level, or activity of one or more ofNKX22, AHR, EGR1, EGR2, EGR3, NGFIC and/or Delta EF1 conditionalknockout transgenic animal.

In some embodiments, the ability of a test compound to modulate, e.g.,increase or decrease, e.g., permanently or temporarily, expression fromone or more of NKX22, AHR, EGR1, EGR2, EGR3, NGFIC, and/or Delta EF1promoter can be evaluated by, e.g., a routine reporter (e.g., LacZ orGFP) transcription assay. For example, a cell or transgenic animal whosegenome includes a reporter gene operably linked to an NKX22, AHR, EGR1,EGR2, EGR3, NGFIC and/or Delta EF1 promoter can be contacted with a testcompound; the ability of the test compound to increase or decrease theactivity of the reporter gene or gene product is indicative of theability of the compound to modulate expression of the TF. In anotherexample, a cell or transgenic animal whose genome includes a reportergene operably linked to a promoter comprising a recognition sequence forone of those TFs, e.g., all or a portion of the Foxp3 promotercomprising recognition sequences for one of those TFs, can be contactedwith a test compound; the ability of the test compound to increase ordecrease the activity of the reporter gene or gene product is indicativeof the ability of the compound to modulate activity of the TF.

The test compound can be administered to a cell, cell extract, explant,or subject (e.g., an experimental animal) expressing a transgenecomprising an NKX22, AHR, EGR1, EGR2, EGR3, NGFIC, and/or Delta EF1promoter or recognition sequence fused to a reporter such as GFP or LacZ(see, e.g., Nehls et al., Science, 272:886-889 (1996), and Lee et al.,Dev. Biol., 208:362-374 (1999), describing placing thebeta-galactosidase reporter gene under control of the whn promoter).Enhancement or inhibition of transcription of a transgene, e.g., areporter such as LacZ or GFP, as a result of an effect of the testcompound on the promoter or factors regulating transcription from thepromoter, can be used to assay an effect of the test compound ontranscription of one or more of the TFs identified herein. Reportertranscript levels, and thus promoter activity, can also be monitored byother known methods, e.g., Northern analysis, ribonuclease protectionassay, reverse transcription-polymerase chain reaction (RT-PCR) or RNAin situ hybridization (see, e.g., Cuncliffe et al., Mamm. Genome,13:245-252 (2002); Sambrook et al., Molecular Cloning: A LaboratoryManual, 3^(rd) Ed., Cold Spring Harbor Laboratory Press (2001)). Testcompounds can also be evaluated using a cell-free system, e.g., anenvironment including a promoter-reporter transgene (e.g., an ARNTpromoter-LacZ transgene), transcription factors binding the promoter, acrude cell lysate or nuclear extract, and one or more test compounds(e.g., a test compound as described herein), wherein an effect of thecompound on promoter activity is detected as a color change.

In one embodiment, the screening methods described herein include theuse of a chromatin immunoprecipitation (ChIP) assay, in which cells,e.g., pancreatic beta cells, expressing one or more of the TFsidentified herein, are exposed to a test compound. The cells areoptionally subjected to crosslinking, e.g., using UV or formaldehyde, toform DNA-protein complexes, and the DNA is fragmented. The DNA-proteincomplexes are immunoprecipitated, e.g., using an antibody directed toone or more of the TFs identified herein. The protein is removed (e.g.,by enzymatic digestion) and analyzed, e.g., using a microarray. In thisway, changes in binding of the transcription factor to its target genescan be evaluated, thus providing a measure of activity of the TFsidentified herein.

Test Compounds

Test compounds for use in the methods described herein are not limitedand can include crude or partially or substantially purified extracts oforganic sources, e.g., botanical (e.g., herbal) and algal extracts,inorganic elements or compounds, as well as partially or substantiallypurified or synthetic compounds, e.g., small molecules, polypeptides,antibodies, and polynucleotides, and libraries thereof.

A test compound that has been screened by a method described herein anddetermined to increase expression, levels, or activity of one or more ofthe TFs described herein can be considered a candidate compound for thetreatment of a disorder treatable with immune therapy (i.e., byincreasing or decreasing control of the immune response by increasing ordecreasing levels of Treg), e.g., cancer, or an autoimmune disorder. Acandidate compound that has been screened, e.g., in an in vivo model ofa disorder treatable with immune therapy, e.g., cancer, or an autoimmunedisorder, and determined to have a desirable effect on the disorder,e.g., on one or more symptoms of the disorder, can be considered acandidate therapeutic agent. Candidate therapeutic agents, once screenedand verified in a clinical setting, are therapeutic agents. Candidatetherapeutic agents and therapeutic agents can be optionally optimizedand/or derivatized, and formulated with physiologically acceptableexcipients to form pharmaceutical compositions.

Methods of Treatment

As described above, the present invention is based, at least in part, onthe identification of useful targets for therapeutic immunomodulation.Accordingly, the present invention provides compositions and methods fortreating a patient (e.g., a human) with an immunological condition.Immunological conditions that will benefit from treatment using thepresent invention include those diseases or disorders caused by anautoimmune response or an absent or insufficient immune response.

Autoimmunity

Autoimmunity is presently the most common cause of disease in the worldand is the third most prevent disease in the U.S. Autoimmune conditionsthat may benefit from treatment using the compositions and methodsdescribed herein include, but are not limited to, for example, Addison'sDisease, alopecia, ankylosing spondylitis, antiphospholipid syndrome,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmuneoophoritis, Bechet's disease, bullous pemphigoid, celiac disease,chronic fatigue immune dysfunction syndrome (CFIDS), chronicinflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,cicatricial pemphigoid, cold agglutinin disease, CREST Syndrome, Crohn'sdisease, diabetes (e.g., type I), dysautonomia, endometriosis,eosinophilia-myalgia syndrome, essential mixed cryoglobulinemia,fibromyalgia, syndrome/fibromyositis, Graves' disease, Guillain Barrésyndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), inflammatory bowel disease(IBD), lichen planus, lupus, Meniere's disease, mixed connective tissuedisease (MCTD), multiple sclerosis, myasthenia gravis, pemphigus,pernicious anemia, polyarteritis nodosa, polychondritis, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud'sphenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy,stiff-man syndrome, Takayasu arteritis, temporal arteritis/giant cellarteritis, thyroid disease, ulcerative colitis, uveitis, vasculitis,vitiligo, and Wegener's granulomatosis.

As described herein, a patient with one or more autoimmune conditionscan be treated by increasing the number of Treg cells and/or theactivity of Treg cells in the patient using, e.g., a therapeuticallyeffective amount of one or more transcription factors (e.g., aligand-activated transcription factor such as AHR) and/or one or moretranscription factor ligands (e.g., TCDD, tryptamine (TA), and/or2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE)) that are capable of promoting an increase in the expressionand/or activity of Foxp3, and thereby promoting an increase in thenumber or activity of Treg cells in vitro and/or in vivo.

In some embodiments, the methods include administering (e.g., to apopulation of T cells or to a subject) a composition comprising anucleic acid encoding a transcription factor as described herein, e.g.,e.g., NKX22, AHR, EGR1, EGR2, EGR3, NGFIC and/or Delta EF1. The nucleicacid can be in an expression vector, e.g., a modified viral vector suchas is known in the art, e.g., a lentivirus, retrovirus, or adenovirus.Methods for using these vectors in cell or gene therapy protocols areknown in the art. For cell therapy methods, it is desirable to startwith a population of T cells taken from the subject to be treated.

In some embodiments, the methods include administering a compositioncomprising a ligand that activates a transcription factor describedherein, e.g., the AHR receptor. In some embodiments, the ligand isco-administered with one or more inhibitors of its degradation, e.g.,tryptamine together with a monoamine oxidase inhibitor, e.g.,tranylcypromine. The inhibitor can be administered in the same or in aseparate composition. Thus the invention also includes compositionscomprising tryptamine and an inhibitor of its degradation, e.g., a MAOI,e.g., tranylcypromine.

In some embodiments, a patient in need of treatment can be administereda pharmaceutically effective dose of one or more ligands capable ofpromoting an increase in the expression and/or activity of Foxp3 andthereby promoting an increase in the number or activity of Treg cells invitro and/or in vivo (e.g., TCDD, tryptamine (TA), and/or2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE)).

Alternatively or in addition, a population of cells capable ofdifferentiation into Treg cells (e.g., naïve T cells and/or CD4⁺CD62ligand⁺ T cells) can be contacted with a transcription factor ligandcapable of promoting increase in Foxp3 expression and/or activity (e.g.,TCDD, tryptamine (TA), and/or2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE)) in vitro, thereby effectively promoting an increase in the numberof Treg cells in the population. Alternatively or in addition, apopulation of cells containing Treg cells (e.g., isolated Treg cells(e.g., 100%) or a population of cells containing at least 20, 30, 40,50, 60, 70, 80, 90, 95, or 99% Treg cells) can be contacted with atranscription factor ligand capable of promoting an increase in Foxp3expression and/or activity (e.g., TCDD, tryptamine (TA), and/or2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE)), thereby effectively promoting an increase in the activity of theTreg cells in the population. Alternatively or in addition, the cellscan be contacted with an expression vector, e.g., a viral vector such asa lentivirus, retrovirus, or adenovirus, comprising a nucleic acidencoding a transcription factor described herein, e.g., NKX22, AHR,EGR1, EGR2, EGR3, NGFIC and Delta EF1. In some embodiments, the cellsare also activated, e.g., by contacting them with an effective amount ofa T cell activating agent, e.g., a composition of one or both ofanti-CD3 antibodies and anti-CD28 antibodies. One or more cells fromthese populations can then be administered to the patient alone or incombination with one or more ligands capable of promoting an increase inthe expression and/or activity of Foxp3 and thereby promoting anincrease in the number or activity of Treg cells in vitro and/or in vivo(e.g., TCDD, tryptamine (TA), and/or2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE).

Patient Selection

The compositions and methods described herein are of particular use fortreating a patient (e.g., a human) that would benefit from therapeuticimmunomodulation (e.g., a patient in need of a suppressed immuneresponse). The methods include selecting a patient in need of treatmentand administering to the patient one or more of the compositionsdescribed herein. A subject in need of treatment can be identified,e.g., by their medical practitioner.

In some embodiments, the methods include determining presence and/orlevels of autoantibodies to an autoantigen specific for the disease,e.g., the presence and/or levels of autoantibodies to an autoantigenlisted in Table 1 or 2. The results can be used to determine a subject'slikelihood or risk of developing the disease; subjects can be selectedfor treatment using a method described herein based on the presenceand/or levels of autoantibodies.

Validation of Treatment/Monitoring Treatment Efficacy

During and/or following treatment, a patient can be assessed at one ormore time points, for example, using methods known in the art forassessing severity of the specific autoimmune disease or its symptoms,to determine the effectiveness of the treatment. In some embodiments,levels of autoantibodies to an autoantigen specific for the disease canalso be monitored, e.g., levels of autoantibodies to an autoantigenlisted in Table 1 or 2; a decrease (e.g., a significant decrease) inlevels of autoantibodies would indicate a positive response, i.e.,indicating that the treatment is successful; see, e.g., Quintana et al.,Proc. Natl. Acad. Sci. U.S.A., 101(suppl. 2):14615-14621 (2004).Treatment can then be continued without modification, modified toimprove the progress or outcome (e.g., increase dosage levels, frequencyof administration, the amount of the pharmaceutical composition, and/orchange the mode of administration), or stopped.

Administration

A therapeutically effective amount of one or more of the compositionsdescribed herein can be administered by standard methods, for example,by one or more routes of administration, e.g., by one or more of theroutes of administration currently approved by the United States Foodand Drug Administration (FDA; see, for example world wide web addressfda.gov/cder/dsm/DRG/drg00301.htm), e.g., orally, topically, mucosally,intravenously or intramuscularly.

In some embodiments, one or more of the ligands described herein can beadministered orally with surprising effectiveness.

Pharmaceutical Formulations

A therapeutically effective amount of one or more of the compositions(e.g., including, but not limited to, one or more of the small moleculeligands, for example TCDD, tryptamine (TA), and/or2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE)) described herein can be incorporated into pharmaceuticalcompositions suitable for administration to a subject, e.g., a human.Such compositions typically include the composition and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances are known. Except insofaras any conventional media or agent is incompatible with the activecompound, such media can be used in the compositions of the invention.Supplementary active compounds can also be incorporated into thecompositions, e.g., an inhibitor of degradation of the ligand.

In some embodiments, the composition can also include an autoantigen,e.g., an autoantigen listed in Table 1 or 2, or another autoantigenknown in the art to be associated with an autoimmune disease.

A pharmaceutical composition can be formulated to be compatible with itsintended route of administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thecomposition (e.g., an agent described herein) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,PRIMOGEL™ (sodium carboxymethyl starch), or corn starch; a lubricantsuch as magnesium stearate or STEROTES™; a glidant such as colloidalsilicon dioxide; a sweetening agent such as sucrose or saccharin; or aflavoring agent such as peppermint, methyl salicylate, or orangeflavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Nucleic acid molecules can be inserted into vectors and used as genetherapy vectors. Gene therapy vectors can be delivered to a subject by,for example, intravenous injection, local administration (see U.S. Pat.No. 5,328,470) or by stereotactic injection (see e.g., Chen et al., PNAS91:3054-3057, 1994). The pharmaceutical preparation of the gene therapyvector can include the gene therapy vector in an acceptable diluent, orcan include a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. In one aspect,the pharmaceutical compositions can be included as a part of a kit.

Generally the dosage used to administer a pharmaceutical compositionsfacilitates an intended purpose for prophylaxis and/or treatment withoutundesirable side effects, such as toxicity, irritation or allergicresponse. Although individual needs may vary, the determination ofoptimal ranges for effective amounts of formulations is within the skillof the art. Human doses can readily be extrapolated from animal studies(Katocs et al., Chapter 27 In: “Remington's Pharmaceutical Sciences”,18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990).Generally, the dosage required to provide an effective amount of aformulation, which can be adjusted by one skilled in the art, will varydepending on several factors, including the age, health, physicalcondition, weight, type and extent of the disease or disorder of therecipient, frequency of treatment, the nature of concurrent therapy, ifrequired, and the nature and scope of the desired effect(s) (Nies etal., Chapter 3, In: Goodman & Gilman's “The Pharmacological Basis ofTherapeutics”, 9th Ed., Hardman et al., eds., McGraw-Hill, New York,N.Y., 1996).

AHR Ligand-Nanoparticles

As demonstrated herein, compositions comprising nanoparticles linked toAHR ligands are surprisingly effective in delivering the ligand, bothorally and by injection, and in inducing the Treg response in livinganimals. Thus, the invention further includes compositions comprisingAHR ligands linked to biocompatible nanoparticles, optionally withantibodies that target the nanoparticles to selected cells or tissues.

AHR Transcription Factor Ligands

AHR-specific ligands, e.g., the high affinity AHR ligand2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), tryptamine (TA), and/or2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE), promote an increase in the number and/or activity of Tregimmunomodulatory cells, which will be useful to suppress the immuneresponse in the treatment of diseases or disorders caused by an abnormal(e.g., an excessive, elevated, or inappropriate) immune response, e.g.,an autoimmune disease or disorder.

Other potentially useful AHR transcription factor ligands are describedin Denison and Nagy, Ann. Rev. Pharmacol. Toxicol., 43:309-34, 2003, andreferences cited herein, all of which are incorporated herein in theirentirety. Other such molecules include planar, hydrophobic HAHs (such asthe polyhalogenated dibenzo-pdioxins, dibenzofurans, and biphenyls) andPAHs (such as 3-methylcholanthrene, benzo(a)pyrene, benzanthracenes, andbenzoflavones), and related compounds. (Denison and Nagy, 2003, supra).Nagy et al., Toxicol. Sci. 65:200-10 (2002), described a high-throughputscreen useful for identifying and confirming other ligands. See alsoNagy et al., Biochem. 41:861-68 (2002). In some embodiments, thoseligands useful in the nanoparticle compositions are those that bindcompetitively with TCDD, TA, and/or ITE.

Biocompatible Nanoparticles

The nanoparticles useful in the methods and compositions describedherein are made of materials that are (i) biocompatible, i.e., do notcause a significant adverse reaction in a living animal when used inpharmaceutically relevant amounts; (ii) feature functional groups towhich the binding moiety can be covalently attached, (iii) exhibit lownon-specific binding of interactive moieties to the nanoparticle, and(iv) are stable in solution, i.e., the nanoparticles do not precipitate.The nanoparticles can be monodisperse (a single crystal of a material,e.g., a metal, per nanoparticle) or polydisperse (a plurality ofcrystals, e.g., 2, 3, or 4, per nanoparticle).

A number of biocompatible nanoparticles are known in the art, e.g.,organic or inorganic nanoparticles. Liposomes, dendrimers, carbonnanomaterials and polymeric micelles are examples of organicnanoparticles. Quantum dots can also be used. Inorganic nanoparticlesinclude metallic nanoparticle, e.g., Au, Ni, Pt and TiO2 nanoparticles.Magnetic nanoparticles can also be used, e.g., spherical nanocrystals of10-20 nm with a Fe2+ and/or Fe3+ core surrounded by dextran or PEGmolecules. In some embodiments, colloidal gold nanoparticles are used,e.g., as described in Qian et al., Nat. Biotechnol. 26(1):83-90 (2008);U.S. Pat. Nos. 7,060,121; 7,232,474; and U.S. P.G. Pub. No.2008/0166706. Suitable nanoparticles, and methods for constructing andusing multifunctional nanoparticles, are discussed in e.g., Sanvicensand Marco, Trends Biotech., 26(8): 425-433 (2008).

In all embodiments, the nanoparticles are attached (linked) to the AHRligands described herein via a functional groups. In some embodiments,the nanoparticles are associated with a polymer that includes thefunctional groups, and also serves to keep the metal oxides dispersedfrom each other. The polymer can be a synthetic polymer, such as, butnot limited to, polyethylene glycol or silane, natural polymers, orderivatives of either synthetic or natural polymers or a combination ofthese. Useful polymers are hydrophilic. In some embodiments, the polymer“coating” is not a continuous film around the magnetic metal oxide, butis a “mesh” or “cloud” of extended polymer chains attached to andsurrounding the metal oxide. The polymer can comprise polysaccharidesand derivatives, including dextran, pullanan, carboxydextran,carboxmethyl dextran, and/or reduced carboxymethyl dextran. The metaloxide can be a collection of one or more crystals that contact eachother, or that are individually entrapped or surrounded by the polymer.

In other embodiments, the nanoparticles are associated withnon-polymeric functional group compositions. Methods are known tosynthesize stabilized, functionalized nanoparticles without associatedpolymers, which are also within the scope of this invention. Suchmethods are described, for example, in Halbreich et al., Biochimie, 80(5-6):379-90, 1998.

In some embodiments, the nanoparticles have an overall size of less thanabout 1-100 nm, e.g., about 25-75 nm, e.g., about 40-60 nm, or about50-60 nm in diameter. The polymer component in some embodiments can bein the form of a coating, e.g., about 5 to 20 nm thick or more. Theoverall size of the nanoparticles is about 15 to 200 nm, e.g., about 20to 100 nm, about 40 to 60 nm; or about 60 nm.

Synthesis of Nanoparticles

There are varieties of ways that the nanoparticles can be prepared, butin all methods, the result must be a nanoparticle with functional groupsthat can be used to link the nanoparticle to the binding moiety.

For example, AHR ligands can be linked to the metal oxide throughcovalent attachment to a functionalized polymer or to non-polymericsurface-functionalized metal oxides. In the latter method, thenanoparticles can be synthesized according to a version of the method ofAlbrecht et al., Biochimie, 80 (5-6): 379-90, 1998. Dimercapto-succinicacid is coupled to the nanoparticle and provides a carboxyl functionalgroup. By functionalized is meant the presence of amino or carboxyl orother reactive groups that can be used to attach desired moieties to thenanoparticles, e.g., the AHR ligands described herein or antibodies.

In another embodiment, the AHR ligands are attached to the nanoparticlesvia a functionalized polymer associated with the nanoparticle. In someembodiments, the polymer is hydrophilic. In a specific embodiment, theconjugates are made using oligonucleotides that have terminal amino,sulfhydryl, or phosphate groups, and superparamagnetic iron oxidenanoparticles bearing amino or carboxy groups on a hydrophilic polymer.There are several methods for synthesizing carboxy and aminoderivatized-nanoparticles. Methods for synthesizing functionalized,coated nanoparticles are discussed in further detail below.

Carboxy functionalized nanoparticles can be made, for example, accordingto the method of Gorman (see WO 00/61191). Carboxy-functionalizednanoparticles can also be made from polysaccharide coated nanoparticlesby reaction with bromo or chloroacetic acid in strong base to attachcarboxyl groups. In addition, carboxy-functionalized particles can bemade from amino-functionalized nanoparticles by converting amino tocarboxy groups by the use of reagents such as succinic anhydride ormaleic anhydride.

Nanoparticle size can be controlled by adjusting reaction conditions,for example, by varying temperature as described in U.S. Pat. No.5,262,176. Uniform particle size materials can also be made byfractionating the particles using centrifugation, ultrafiltration, orgel filtration, as described, for example in U.S. Pat. No. 5,492,814.

Nanoparticles can also be treated with periodate to form aldehydegroups. The aldehyde-containing nanoparticles can then be reacted with adiamine (e.g., ethylene diamine or hexanediamine), which will form aSchiff base, followed by reduction with sodium borohydride or sodiumcyanoborohydride.

Dextran-coated nanoparticles can also be made and cross-linked, e.g.,with epichlorohydrin. The addition of ammonia will react with epoxygroups to generate amine groups, see Hogemann et al., Bioconjug. Chem.2000. 11(6):941-6, and Josephson et al., Bioconjug. Chem., 1999,10(2):186-91.

Carboxy-functionalized nanoparticles can be converted toamino-functionalized magnetic particles by the use of water-solublecarbodiimides and diamines such as ethylene diamine or hexane diamine.

Avidin or streptavidin can be attached to nanoparticles for use with abiotinylated binding moiety, such as an oligonucleotide or polypeptide.See e.g., Shen et al., Bioconjug. Chem., 1996, 7(3):311-6. Similarly,biotin can be attached to a nanoparticle for use with an avidin-labeledbinding moiety.

In all of these methods, low molecular weight compounds can be separatedfrom the nanoparticles by ultra-filtration, dialysis, magneticseparation, or other means. The unreacted AHR ligands can be separatedfrom the ligand-nanoparticle conjugates, e.g., by size exclusionchromatography.

In some embodiments, colloidal gold nanoparticles are made using methodsknown in the art, e.g., as described in Qian et al., Nat. Biotechnol.26(1):83-90 (2008); U.S. Pat. Nos. 7,060,121; 7,232,474; and U.S. P.G.Pub. No. 2008/0166706.

In some embodiments, the nanoparticles are pegylated, e.g., as describedin U.S. Pat. Nos. 7,291,598; 5,145,684; 6,270,806; 7,348,030, andothers.

Antibodies

In some embodiments, the nanoparticles also include antibodies toselectively target a cell. The term “antibody,” as used herein, refersto full-length, two-chain immunoglobulin molecules and antigen-bindingportions and fragments thereof, including synthetic variants. A typicalfull-length antibody includes two heavy (H) chain variable regions(abbreviated herein as VH), and two light (L) chain variable regions(abbreviated herein as VL). The term “antigen-binding fragment” of anantibody, as used herein, refers to one or more fragments of afull-length antibody that retain the ability to specifically bind to atarget. Examples of antigen-binding fragments include, but are notlimited to: (i) a Fab fragment, a monovalent fragment consisting of theVL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., Nature 341:544-546 (1989)),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. Science 242:423-426 (1988); and Huston et al. Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such single chain antibodiesare also encompassed within the term “antigen-binding fragment.”

Production of antibodies and antibody fragments is well documented inthe field. See, e.g., Harlow and Lane, 1988. Antibodies, A LaboratoryManual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. Forexample, Jones et al., Nature 321: 522-525 (1986), which disclosesreplacing the CDRs of a human antibody with those from a mouse antibody.Marx, Science 229:455-456 (1985), discusses chimeric antibodies havingmouse variable regions and human constant regions. Rodwell, Nature342:99-100 (1989), discusses lower molecular weight recognition elementsderived from antibody CDR information. Clackson, Br. J. Rheumatol. 3052:36-39 (1991), discusses genetically engineered monoclonal antibodies,including Fv fragment derivatives, single chain antibodies, fusionproteins chimeric antibodies and humanized rodent antibodies. Reichmanet al., Nature 332: 323-327 (1988) discloses a human antibody on whichrat hypervariable regions have been grafted. Verhoeyen, et al., Science239: 1534-1536 (1988), teaches grafting of a mouse antigen binding siteonto a human antibody.

In the methods described herein, it would be desirable to target thecompounds to T cells, B cells, dendritic cells, and/or macrophages,therefore antibodies selective for one or more of those cell types canbe used. For example, for T cells, anti-CXCR4, anti-CD28, anti-CD8,anti-TTLA4, or anti-CD3 antibodies can be used; for B cells, antibodiesto CD20, CD19, or to B-cell receptors can be used; for dendritic celltargeting, exemplary antibodies to CD11c, DEC205, MHC class I or classII, CD80, or CD86 can be used; for macrophages, exemplary antiboduies toCD11b, MHC class I or class II, CD80, or CD86 can be used. Othersuitable antibodies are known in the art.

Kits

The present invention also includes kits. In some embodiments the kitcomprise one or more doses of a composition described herein. Thecomposition, shape, and type of dosage form for the induction regimenand maintenance regimen may vary depending on a patients requirements.For example, dosage form may be a parenteral dosage form, an oral dosageform, a delayed or controlled release dosage form, a topical, and amucosal dosage form, including any combination thereof.

In a particular embodiment, a kit can contain one or more of thefollowing in a package or container: (1) one or more doses of acomposition described herein; (2) one or more pharmaceuticallyacceptable adjuvants or excipients (e.g., a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, and clathrate); (3) one or morevehicles for administration of the dose; (5) instructions foradministration. Embodiments in which two or more, including all, of thecomponents (1)-(5), are found in the same container can also be used.

When a kit is supplied, the different components of the compositionsincluded can be packaged in separate containers and admixed immediatelybefore use. Such packaging of the components separately can permit longterm storage without loosing the active components' functions. When morethan one bioactive agent is included in a particular kit, the bioactiveagents may be (1) packaged separately and admixed separately withappropriate (similar of different, but compatible) adjuvants orexcipients immediately before use, (2) packaged together and admixedtogether immediately before use, or (3) packaged separately and admixedtogether immediately before use. If the chosen compounds will remainstable after admixing, the compounds may be admixed at a time before useother than immediately before use, including, for example, minutes,hours, days, months, years, and at the time of manufacture.

The compositions included in particular kits of the present inventioncan be supplied in containers of any sort such that the life of thedifferent components are optimally preserved and are not adsorbed oraltered by the materials of the container. Suitable materials for thesecontainers may include, for example, glass, organic polymers (e.g.,polycarbonate and polystyrene), ceramic, metal (e.g., aluminum), analloy, or any other material typically employed to hold similarreagents. Exemplary containers may include, without limitation, testtubes, vials, flasks, bottles, syringes, and the like.

As stated above, the kits can also be supplied with instructionalmaterials. These instructions may be printed and/or may be supplied,without limitation, as an electronic-readable medium, such as a floppydisc, a CD-ROM, a DVD, a Zip disc, a video cassette, an audiotape, and aflash memory device. Alternatively, instructions may be published on ainternet web site or may be distributed to the user as an electronicmail.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1: Cloning and Characterization of Zebrafish Foxp3

The zebrafish is an experimental model of vertebrate development; asdescribed herein, it can also be used as an immunogenic model. Thisexample describes the cloning and characterize of the zebrafish (Daniorerio) functional homologue of mammalian Foxp3 (herein termed zFoxp3).

Identification of zFoxp3

To investigate whether Foxp3-dependent immunoregulatory mechanismsoperate in the zebrafish, we searched the zebrafish genome for a Foxp3homologue, which we termed zFoxp3 (FIG. 1E). A phylogenetic analysisplaced zFoxp3 in a sub-tree together with mammalian and other fishorthologous predictions, suggesting that zFoxp3 is the zebrafishortholog for mammalian Foxp3 (FIG. 1F). In mammals Foxp3 is located in awell-conserved synteny block. Indeed, we found several orthologous genesbetween mammalian chromosome X and zebrafish chromosome 8 in the regionwhere zFoxp3 is located (suv39h1, cacna1s, tspyl2, wasp), strengtheningthe likelihood of zFoxp3 being the fish ortholog of Foxp3.

The accession numbers for the amino acid sequences used in the gene treeanalysis are as follows: Danio rerio Foxp1a Q08BX8 BC124513; Foxp1bQ2LE08 NM_001039637; Foxp2 Q4JNX5 NM_001030082; Foxp3 annotated (ESTCK028390); Foxp4 annotated. Homo sapiens: Foxp1 Q9H334 NM_001012505,Foxp2 O15409 NM_148899, Foxp3 Q9BZS 1 NM_014009, Foxp4 Q8IVH2 NM_138457;Mus musculus: Foxp1 P58462 NM_053202, Foxp2 P58463 NM_053242, Foxp3Q99JB6 NM_054039, Foxp4 Q9DBY0 NM_028767; Ciona intestinalis FoxpQ4H3H6. The amino acid sequence of the apparent stickleback orthologuesof Foxp1, Foxp2, Foxp3 and Foxp4 were obtained from Ensembl.

Cloning zFoxp3

zFoxp3 was cloned from cDNA prepared from zebrafish kidney by using aTOPO® PCR cloning kit (Invitrogen, CA, USA) according to themanufacturer's instructions.

Characterization of Foxp3

The amino acids (aa) predicted to mediate the interaction of theforkhead domain with DNA (Stroud et al., Structure. 14, 159-66 (2006))or the transcription factor NFAT (Wu et al., Cell 126, 375-87 (2006)) inmammalian Foxp3 are conserved in zFoxp3, as well as aa found to bemutated in humans with impaired Foxp3 activity (Ziegler, Annu RevImmunol. 24, 209-26 (2006)) (FIG. 1E). The zinc finger/leucine zipperdomain is important for the homodimerization of Foxp3 and itstranscriptional regulatory activities (Chae et al., Proc Natl Acad SciUSA 103, 9631-6 (2006)). To study the ability of zFoxp3 to dimerize, wedesigned a pull-down assay using His-tagged zFoxp3 and a renillaluciferase-tagged Foxp3 (Foxp3-Ren). 293 cells were transfected asdescribed (Bettelli et al., Proc Natl Acad Sci USA 102, 5138-43 (2005))and the cells were analyzed after 24 or 48 hours with the dualluciferase assay kit (New England Biolabs, Ipswich, Mass.) (cells werelysed, and zFoxp3 was pulled-down with Ni-Agarose and the renillaluciferase activity in the pellet was quantified). Tk-Renilla was usedfor standardization. Alternatively, the transfected cells were lysed andimmuno-precipitation was carried out as described (Bettelli et al., ProcNatl Acad Sci USA 102, 5138-43 (2005)); hemagglutinin (HA) labeled NFATand NF-kB were detected with anti-HA and anti-P65 antibodies obtainedfrom Santa Cruz Biotechnology (Santa Cruz, Calif., USA).

As shown in FIG. 1G, zFoxp3 pulled-down Foxp3-Ren indicating that zFoxp3can homodimerize. Hence, zFoxp3 has structural features common tomammalian Foxp3.

Foxp3 can physically interact with NF-kB and NFAT to down-regulate theirtranscriptional activities (Wu et al., (2006), supra; Bettelli et al.,(2005), supra). As shown in FIG. 2a , zFoxp3 interfered with theactivation of NFAT and NF-κB responsive promoters. This effect wasstronger for NF-κB. Co-immunoprecipitation experiments showed thatzFoxp3 interacts both with NF-κB and NFAT. In agreement with the reducedinhibitory effect of Foxp3 on NFAT-driven reporters (see FIG. 2a ), thezFoxp3-NFAT interaction was weaker (see FIG. 2b ). These results suggestthat zFoxp3 can directly interact with NFAT and NF-κB to interfere withtheir transcriptional activities.

MSCV GFP-RV retroviral DNA plasmids were transfected into the Phoenixpackaging cell line and 72 hours later the retrovirus-containingsupernatants were collected. MACS-purified CD4+ T cells were activated24 hours later with plate-bound antibodies to CD3 and CD28, and infectedby centrifugation (45 minutes at 2000 rpm) with retrovirus-containingsupernatant supplemented with 8 μg/ml Polybrene (Sigma-Aldrich) andrecombinant human IL-2 (25 units/ml).

Cells were cultured in serum-free X-VIVO 20™ media (BioWhittaker,Walkersville, Md., USA) for 72 hours. During the last 16 hours, cellswere pulsed with 1 μCi of [³H]thymidine (PerkinElmer, Waltham, Mass.,USA) followed by harvesting on glass fiber filters and analysis ofincorporated [³H]thymidine in a beta-counter (1450 Microbeta, Trilux,PerkinElmer). Alternatively, culture supernatants were collected 48after activation and the cytokine concentration was determined by ELISAusing antibodies for IFN-γ, IL-17, IL-4, IL-10 from BD Biosciences andantibodies to TGF-β from R&D Systems. For suppression assays, MACSpurified CD4⁺CD25⁻ T cells from naïve C57BL/6 mice (1-5×10⁴ cells/well)were stimulated with antibodies to CD3 and C57BL/6 irradiated spleencells (0.3-1.5×10⁴ cells/well) for 3 days in the presence of differentratios of CD4⁺GFP⁺ retrovirus-transduced T cells.

Retroviral transduction of zFoxp3 into mouse T cells led to theup-regulation of surface molecules associated with Treg function such asCD25, CTLA-4 and GITR (see FIG. 2c ). Moreover, ectopic expression ofzFoxp3 in mouse T cells led to a significant decrease in theirproliferation and cytokine secretion upon activation with antibodies toCD3 (see FIG. 2d ). Moreover, zFoxp3 transduced T cells could inhibitthe activation of other T cells, both in terms of T cell proliferationand of cytokine secretion, in a dose dependent manner (see FIG. 2e ). Insummary, expression of zFoxp3 in mouse T cells induced a Treg-likephenotype. These data suggest that zFoxp3 is a functional homologue ofmammalian Foxp3, and that Foxp3 is capable of promoting a Treg likephenotype.

Western blot studies of zebrafish tissues identified a Foxp3cross-reactive protein in thymus, kidney and spleen compatible with thepredicted size of zFoxp3.

The expression of zFoxp3 was then analyzed by real-time PCR onFACS-sorted lymphocytes, myelomonocytes and erythrocytes (Traver et al.,Nat Immunol 4, 1238-46 (2003)). RNA was extracted from cells usingRNAeasy columns (Qiagen, Valencia, Calif., USA), complementary DNA wasprepared as recommended (Bio-Rad Laboratories, Hercules, Calif., USA)and used as template for real time PCR. The expression of Foxp3 wasquantified with specific primers and probes (Applied Biosystems, FosterCity, Calif., USA) on the GeneAmp 5500 Sequence Detection System(Applied Biosystems). Expression was normalized to the expression of thehousekeeping gene, GAPDH.

As shown in FIG. 3a , zFoxp3 expression was restricted to the lymphocytefraction. This observation is consistent with the expression pattern ofmammalian Foxp3 and supports the conservation of the regulatorymechanisms of gene expression that control tissue specificity.

Example 2: Identification of Transcription Factor Binding Sites in Foxp3

The elements regulating gene expression in genomic DNA are underselective pressure, and therefore are more conserved than thesurrounding nonfunctional sequences. Phylogenetic footprinting is amethod based on the analysis of sequence conservation betweenorthologous genes from different species to identify regions of DNAinvolved in the regulation of gene expression. Once identified, theseconserved regions can be analyzed with TFBS detection algorithms togenerate a list of putative TFBS.

We performed a phylogenetic footprinting analysis aimed at identifyingregulatory regions within the zebrafish, mouse and human Foxp3 gene(Ovcharenko et al., Genome Res 15, 184-94 (2005)). The inclusion ofdistant species like the zebrafish is highly informative because itfacilitates the identification of conserved regulatory sequences amidstDNA regions that were not subjected to any selective pressure(Ovcharenko et al., Genome Res 15, 184-94 (2005)).

The Mulan server (mulan.dcode.org) was used to perform a phylogeneticfootprinting analysis of Foxp3. Mulan brings together differentalgorithms in a web-based user-friendly interface: programs for therapid identification of local sequence conservation connected to themultiTF/TRANSFAC database for the detection of evolutionarily conservedTFBS in multiple alignments. FIGS. 7A-B show the results obtained usingthe sequences of Foxp3 in rat, mouse, dog, human and zebrafish: PutativeTFBS were found for 6 transcription factors, all of them known to beexpressed and functional in T cells: NKX22, AHR, EGR1, EGR2, EGR3, NGFICand Delta EF1. These TF identified by phylogenetic footprinting areother potential regulators of Foxp3 expression and Treg development.

Example 3: Adaptive Cellular Immunity and Foxp3-DependentImmunoregulation in Zebrafish

The adaptive cellular immune response of 6 month old zebrafish immunizedintraperitoneally (ip) with heat killed M. tuberculosis (MT) or PBS inincomplete Freund's adjuvant (IFA) was studied.

As shown in FIG. 1A, spleen cells prepared 14 days after immunizationwith MT or PBS proliferated in response to stimulation with ConcanavalinA (ConA), but only cells taken from MT-immunized fish proliferated uponactivation with MT.

Another group of six month old zebrafish were anesthetized with 0.02%tricaine (Sigma-Aldrich) and immunized i.p. with 10 μl/fish of zebrafishbrain homogenate (zCNS) emulsified in complete Freund's adjuvant (CFA).As shown in FIGS. 1b-d , this resulted in the accumulation of CD3, IFNgand IL-17 expressing cells in the brain.

These results demonstrated that zebrafish can mount adaptiveantigen-specific cell-mediated immune and autoimmune responses.

C. elegans and D. melanogaster have been extremely useful for theidentification of the genes governing innate immunity (Lemaitre et al.,Nat Rev Immunol 4, 521-7 (2004)). These experimental models, however,lack an adaptive immune system and therefore cannot be used to studyvertebrate-specific immune processes. The zebrafish harbors both innateand adaptive immune systems with functional macrophages (Davis et al.,Immunity 17, 693-702 (2002)), B cells (Danilova et al., Proc Natl AcadSci USA 99, 13711-6 (2002)) and T cells (Danilova et al., Dev CompImmunol 28, 755-67 (2004); Langenau et al., Proc Natl Acad Sci USA 101,7369-74 (2004)). Taking together the presence of basic components of theadaptive immune system (Langenau et al., Nat Rev Immunol. 5, 307-17(2005)) with the experimental advantages offered by the zebrafish forthe realization of large scale genetic and chemical screens (Lieschke etal., Nat Rev Genet. 8, 353-67 (2007)), the zebrafish can serve as anexperimental model for the study of pathways controlling adaptive immuneprocesses such as Treg development.

Example 4: AHR Controls Foxp3 Expression and Treg Generation

Using the methods described above, a conserved binding site for the arylhydrocarbon receptor (AHR) was identified in the genomic sequence ofFoxp3 (see FIGS. 3b and 3j ), which was termed the conserved AHR bindingsite (CABS). A similarly located regulatory sequence controls theexpression of the AHR-regulated cytochrome P4501A2 (CYP1A2). Inaddition, three non-evolutionary conserved AHR-binding sites (NCABS)were identified in the zFoxp3 promoter (termed NCABS-1, -2, and -3) (seeFIGS. 3i and 3j ).

First, Foxp3 expression was measured in mouse Treg isolated fromFoxp3^(gpf) knock in mice. Foxp3^(gpf) knock in mice have a GFP reporterinserted in the Foxp3 gene, producing GFP in Foxp3⁺ Treg, whichfacilitates the identification and FACS sorting of GFP:Foxp3⁺ Treg(Bettelli et al., Nature 441, 235-8 (2006)).

CD4+ T cells were purified from Foxp3gfp knock in mice using anti-CD4beads (Miltenyi, Auburn, Calif., USA) and sorted (FACSAria™ cell sorter,BD Biosciences) into naive CD4⁺Foxp3:GFP⁻ or CD4⁺Foxp3:GFP T cells.CD4⁺Foxp3:GFP⁻ T cells were stimulated with plate bound 1 μg/ml ofanti-CD3 (145-2C11, eBioscience) and 2 μg/ml of anti-CD28 (37.51,eBioscience) for 5 days, supplemented with recombinant IL-2 (50 U/ml) atday 2 and 4, and analyzed by FACS at day 5 for their differentiationinto CD4⁻Foxp3:GFP⁺ Treg. TGFβ1 (2.5 ng/ml) was used as a positivecontrol.

Higher levels of AHR expression were detected on FACS-sortedCD4⁺GFP:Foxp3+ Treg than in CD4⁺GFP:Foxp3⁻ T cells (see FIG. 3c ),highlighting a possible link between AHR and Foxp3 expression. Therelationship between AHR and Foxp3 was then further analyzed usingRT-PCR.

Briefly, CD4+ T cells were purified from Foxp3gfp knock in mice asdescribed above. RNA was then extracted using RNAeasy columns (Qiagen,Valencia, Calif., USA). Complementary DNA was prepared as recommended(Bio-Rad Laboratories, Hercules, Calif., USA) and used as template forreal time PCR. The expression of Foxp3 was quantified with specificprimers and probes (Applied Biosystems, Foster City, Calif., USA) on theGeneAmp 5500 Sequence Detection System (Applied Biosystems). Expressionwas normalized to the expression of the housekeeping genes, GAPDH oractin.

Data generated using RT-PCR corroborated the observed associationbetween AHR and Foxp3. In addition, CYP1A1 expression, a AHR responsivegene, was also observed in CD4⁺GFP:Foxp3⁺ Treg cells. Furthermore, asshown in FIG. 3n , treatment of the cells with the AHR antagonistresveratrol resulted in a significant decrease in both Foxp3 and CYP1A1expression levels (P<0.0023 and P<0.0235, respectively). Decreases inthe suppressive activity was also noted in resveratrol treated cells(FIG. 3o ). Together, these results strongly suggest that the detectedAHR is functional.

To investigate whether AHR directly controls Foxp3 expression, we used abacterial artificial chromosome that contained the entire foxp3 locustagged with a Renilla luciferase reporter after the ATG start codon.More specifically, we used the RP23-267C15 BAC clone, which contains 200kb of mouse genomic DNA, including the entire locus of the Foxp3 gene. ARenilla cDNA cassette was the cloned immediately after the ATG startcodon of Foxp3 gene by homologous recombination using the Redrecombineering system contained in the DY 380 bacteria strain. The finalconstruct was designated BACFoxp3:Ren.

As shown in FIG. 3k , cotransfection of BACFoxp3:Ren with a constructcoding for mouse AHR resulted in a significant up-regulation of Renillaactivity (P<0.01), similar to that achieved with a constitutivelyactivated TGFβ receptor II. This observation demonstrates that AHR iscapable of directly controlling Foxp3 expression.

Chromatin immunoprecipitation (ChIP) was then applied to analyze theinteraction of AHR with the CABS and NCABS shown in FIGS. 3b and 3i ,respectively.

Briefly, cells were treated for 90′ with TCDD, fixed with 1%formaldehyde for 15 minutes and quenched with 0.125 M glycine. Chromatinwas isolated and sheared to an average length of 300-500 bp bysonication. Genomic DNA (input) was prepared by treating aliquots ofchromatin with RNase, proteinase K and heat for de-crosslinking,followed by ethanol precipitation. AHR-bound DNA sequences wereimmuno-precipitated with an AHR-specific antibody (Biomol SA-210).Crosslinks were reversed by incubation overnight at 65° C., and ChIP DNAwas purified by phenol-chloroform extraction and ethanol precipitation.Quantitative PCR reactions were then performed using the followingprimer pairs:

Cyp1a1-845 F: aggctcttctcacgcaactc (SEQ ID NO: 144) and Cyp1a1-845 R:ctggggctacaaagggtgat (SEQ ID NO: 145);

Foxp3 (NCAB-1)−2269 F: agctgcccattacctgttag (SEQ ID NO: 146) and Foxp3(NCAB-1)−2269 R: ggaggtctgcatggatcttag (SEQ ID NO:147);

Foxp3 (NCAB-2)−1596 F: gccttgtcaggaaaaactctg (SEQ ID NO: 148) and Foxp3(NCAB-2)−1596 R: gtcctcgatttggcacagac (SEQ ID NO: 149);

Foxp3 (NCAB-3)−800 F: cttgcccttcttggtgatg (SEQ ID NO: 150) and Foxp3(NCAB-3)−800 R: ttgtgctgagtgccctgac (SEQ ID NO:151);

Foxp3 (CAB)+13343 F: gctttgtgcgagtggagag (SEQ ID NO:152) and Foxp3(CAB)+13343 R: agggattggagcacttgttg (SEQ ID NO: 153).

The Untr6 region in chromosome 6 located at chr6:120, 258, 582-120, 258,797 was amplified as a control using Untr6 F: tcaggcatgaaccaccatac (SEQID NO: 154) and Untr6 R: aacatccacacgtccagtga (SEQ ID NO: 155).

Experimental Ct values were converted to copy numbers detected bycomparison with a DNA standard curve run on the same PCR plates. Copynumber values were then normalized for primer efficiency by dividing bythe values obtained using Input DNA and the same primer pairs. Errorbars represent standard deviations calculated from the triplicatedeterminations.

ChIP analysis of the interaction of AHR with CABS and NCABS in Foxp3 andCYP1A1 was then performed in control and2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; a high affinity AHR ligand)treated CD4⁺ T cells and T cells isolated from mice.

As shown in FIG. 31, treatment of CD4+ T cells with TCDD increased AHRbinding to the CABS and NCABS-2 (p<0.05). This up-regulation wascomparable to that detected in the promoter of the AHR-regulated genecytochrome P4501A1 (CYP1A1) gene. No significant increase in AHR bindingwas seen in NCABS-1, NCABS-3 or the control sequence UTR6. As shown inFIG. 3m , similar results were obtained when CD4 T cells were purifiedfrom TCDD treated mice. These data suggest that AHR controls Foxp3expression.

TCDD was also used to characterize the functional relationship betweenAHR and Foxp3. Treatment of 3-day post-fertilization zebrafish embryoswith TCDD led to a dose-dependent increase in zFoxp3 expression,suggesting that the conserved AHR binding site in the zFoxp3 sequence isfunctional (see FIG. 3D).

We then studied the effect of AHR activation on mouse Treg numbers.Naïve C57BL/6 mice were treated with TCDD (1 mg/mouse, ip) and immunized24 hours later with MOG₃₅₋₅₅ in CFA. Draining lymph nodes were prepared10 days later and CD4⁺Foxp3⁺ Treg were quantified by FACS.Administration of TCDD led to a small increase in the number ofCD4⁺Foxp3⁺ Treg (see FIG. 3E). Moreover, a single administration of TCDDfollowed by immunization with MOG₃₅₋₅₅ led to a significant increase inthe number of the CD4⁺Foxp3⁺ T cells (see FIG. 3E). Furthermore, theCD4⁺Foxp3:GFP⁺ T cells expanded in vivo by TCDD administration andMOG₃₅₋₅₅ immunization were functional and showed increasedMOG₃₅₋₅₅-specific suppressive activity (see FIG. 3P).

To rule out any direct cytotoxic or pro-apoptotic effect of TCDD oneffector T cells, purified mouse CD4⁺CD25⁻ T cells were activated invitro with antibodies to CD3 in the presence of TCDD. Incubation withTCDD did not increase T cell apoptosis as measured by annexin-FITCstaining and did not decrease the proliferative response (see FIG. 3F).Taken together, these data suggest that AHR controls Foxp3 expressionand Treg expansion both in zebrafish and in mice.

To establish if TCDD triggered the conversion of CD4⁺Foxp3⁻ T cells intonew Foxp3⁺ Treg cells, FACS sorted CD4⁺Foxp3:GFP− T cells were activatedin vitro with antibodies to CD3 and CD28 in the presence of TCDD, andthe generation of CD4⁺Foxp3:GFP⁺ Treg was followed by FACS. TGFb1 wasused as a positive control. As shown in FIG. 3G, TCDD triggered theconversion of approximately 13% of the cells in culture intoCD4⁺Foxp3:GFP⁺ Treg. Additionally, as shown in FIG. 3, CD4⁺Foxp3:GFP⁺Treg induced by TCDD showed a suppressive activity similar to that ofTreg induced in vitro with TGFβ1 or CD4⁺Foxp3:GFP⁺ Treg sorted fromnaïve Foxp3gpf mice.

Thus, AHR activation by the high affinity AHR ligand TCDD can triggerthe conversion of CD4⁺Foxp3⁻ T cells into functional CD4⁺GFP⁺ Treg.

As shown in FIGS. 3r and 3s , treatment with the AHR antagonistresveratrol (50 μM) interfered with the induction of Treg by TGFβ1 andTCDD, but had a stronger effect on the Treg conversion triggered by TCDD(P=0.0053, FIG. 1h ). CD4⁺Foxp3:GFP⁺ Treg purified from naïve mice didnot proliferate and did not show increased suppressive activity uponstimulation with antibodies to CD3 and CD28 and TCDD. These observationssuggest that AHR is more important for the differentiation of new Tregthan for the activity of established Treg.

To investigate if new Treg could also be generated in vivo followingTCDD administration, we transferred CD4⁺Foxp3:GFP⁻ 2D2 T cells fromCD90.2 donors into wild type CD90.1 recipients. CD4⁺Foxp3:GFP⁻ 2D2 Tcells express a MOG₃₅₋₅₅-specific T cell receptor. The recipients wereadministered 1 μg/mouse TCDD and were immunized 2 days later withMOG₃₅₋₅₅. CD4⁺Foxp3:GFP⁺CD90.2 T cells (donor cells that underwentconversion into Treg upon treatment with TCDD) were then quantified byFACS. As shown in FIG. 3h , TCDD promoted a significant (P<0.02,unpaired t-test, n=5) conversion of CD4⁺Foxp3:GFP⁻ CD90.2 donor T cellsinto Treg cells.

Thus, the increase in the frequency of Treg that follows activation ofAHR with TCDD is due, at least in part, to the conversion ofCD4⁺Foxp3:GFP⁻ T cells into CD4⁺Foxp3: GFP⁺ Treg.

Example 5: AHR Activation by TCDD Suppresses EAE

To analyze the functionality of the Treg cells induced by AHRactivation, we studied the effect of TCDD on EAE development.

C57BL/6 mice were given a single intraperitoneal (ip) dose of TCCD, andone day later EAE was induced by immunization with MOG₃₅₋₅₅ in CFA. TCDDwas also administered orally (1 μg/mouse) to determined whether aneffective dose of this ligand can be delivered via oral administrationand whether this dose is capable of reducing EAE development. EAE wasinduced by injecting the mice subcutaneously with 100 ml of the MOG₃₅₋₅₅peptide (MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 156)) in complete Freundadjuvant oil. In addition, the mice received 150 ng of pertussis toxin(Sigma-Aldrich) ip on days 0 and 2. Clinical signs of EAE were assessedaccording to the following score: 0, no signs of disease; 1, loss oftone in the tail; 2, hind limb paresis; 3, hind limb paralysis; 4,tetraplegia; 5, moribund.

As shown in FIG. 4a and Table 3, ip administered TCDD had adose-dependent effect on the clinical signs of EAE. 1 μg/mouse markedlyinhibited the clinical signs of EAE (P<0.001; n=6).

TABLE 3 TCDD Treatment Suppresses EAE Mean day of onset Mean maximumTreatment Incidence (Mean ± standard score (μg per mouse)(positive/total) deviation (SD)) (mean ± SD) Control 42/49 (87%) 13.6 ±2.8 2.4 ± 1.4 TCDD 1 μg  4/40 (10%)  21.8 ± 1.5*  0.2 ± 0.6* TCDD 0.1 μg  5/5 (100%) 17.0 ± 1.2 3.1 ± 0.5 TCDD 0.01 μg 7/7 14.1 ± 2.9 2.7 ± 0.6Mice treated with corn oil (control) or TCDD (ip) were immunized withMOG₃₅₋₅₅ peptide in CFA and monitored for EAE development. Statisticalanalysis was performed by comparing groups using one-way analysis ofvariance. *P < 0.0001.

As shown in FIGS. 4G-4I, IP administered TCDD also reduced thehistopathological signs of EAE.

In addition, orally administered of 1 μg/mouse of TCDD, one day beforeEAE induction, also prevented EAE development (P<0.001, two-way ANOVA,n=10). This observation suggests that an effective does of TCDD can beadministered orally.

To confirm that the effects on EAE were mediated by the activation ofAHR, we used C57BL/6 mice carrying the d allele of the ahr gene (AHR-dmice). This allele codes for a mutant AHR with a 10 fold reduction inits affinity for TCDD and other ligands (Okey et al., Mol Pharmacol. 35,823-30 (1989)) due to mutations in its ligand binding sites. Theadministration of TCDD (1 μg/mouse) to AHR-d mice did not increase thelevels of CD4⁺Foxp3⁺ Treg in AHR-mt mice, and did not inhibit theprogression of EAE, as shown in FIG. 4B and Table 4.

TABLE 4 TCDD Treatment of AHR-d Mice Incidence Mean day of onset Meanmaximum Treatment (positive/total) (Mean ± SD) score (mean ± SD) WTcontrol 12/14 (86%) 13.9 ± 1.9 2.4 ± 1.4 AHR-d + TCDD  9/11 (82%) 17.3 ±3.0^(†) 2.2 ± 1.4 WT + TCDD 1/10 (10%)^(#) 21  0.2 ± 0.6* C57BL/6 (WT)and AHR-d mice treated with corn oil (control) or TCDD (1 μg/mouse) wereimmunized with MOG₃₅₋₅₅ peptide in CFA and monitored for EAEdevelopment. Statistical analysis was performed by comparing groupsusing one-way analysis of variance. *P < 0.001 vs WT control group and P< 0.01 vs AHR-d TCDD group; ^(†)P = 0.0046 vs WT control group; ^(#)P =0.0005 vs WT control group, P = 0.0019 vs AHR-d TCDD group.

Taken together, these results show that TCDD-dependent AHR activationcan inhibit or suppress the development and/or progression of EAE. Thedata presented in Example 3 indicate that this effect is due to theTCDD-dependent AHR activation that promotes the induction of functionalTreg.

Antigen microarrays were then used to study the antibody response tomyelin in mice that did not develop EAE as consequence of AHR activationby TCDD. The antigens listed in Table 1 were spotted onto Epoxy slides(TeleChem, Sunnyvale, Calif., USA) as described (Quintana et al., ProcNatl Acad Sci USA 101 Suppl 2, 14615-21 (2004)). Antigens were spottedin replicates of 6, the microarrays were blocked for 1 h at 37° C. with1% bovine serum albumin, and incubated for 2 hours at 37° C. with a1:100 dilution of the test serum in blocking buffer. The arrays werethen washed and incubated for 45 min at 37° C. with goat anti-mouse IgGCy3-conjugated detection antibodies (Jackson ImmunoResearch Labs, WestGrove, Pa., USA). The arrays were scanned with a ScanArray 4000X scanner(GSI Luminomics, Billerica, Mass., USA). Antigen reactivity was definedby the mean intensity of binding to the replicates of that antigen onthe microarray. Raw data were normalized and analyzed using theGeneSpring software (Silicon Genetics, Redwood City, Calif., USA) withthe non-parametric Wilcoxon-Mann-Whitney test, using the Benjamini andHochberg method with a false discovery rate (FDR) of 0.05 to determinesignificance. The samples were clustered using a pairwise averagelinkage algorithm based on Spearman's rank correlation as a distancemeasure.

The microarrays consisted of a collection of 362 CNS-relatedautoantigens including tissue lysates, recombinant proteins, peptidelibraries spanning the whole sequence of myelin proteins and lipidsfound in the central and peripheral nervous system, a complete list ofthe antigens used is provided in Table 1.

TABLE 1 362 CNS-Related Autoantigens Heat 27 kDa Heat Shock ProteinShock 32 kDa Heat Shock Protein Proteins 40 kDa Heat Shock Protein (HSP)47 kDa Heat Shock Protein 60 kDa Heat Shock Protein 60 kDa Heat ShockProtein peptide: aa 106-125; aa 1-20; aa 121-140; aa 136-155; aa151-170; aa 16-35; aa 166- 185; aa 181-199; aa 195-214; aa 210-229; aa225-244; aa 240-259; aa 255-275; aa 271-290; aa 286-305; aa 301- 320; aa31-50; aa 316-335; aa 331-350; aa 346-365; aa 361-380; aa 376-395; aa391-410; aa 406-425; aa 421- 440; aa 436-455; aa 451-470; aa 466-485; aa46-65; aa 481-500; aa 496-515; aa 511-530; aa 526-545; aa 541- 560; aa556-573; aa 61-80; aa 76-95; or aa 91-110 65 kDa Heat Shock Protein M.tuberculosis 70 kDa Heat Shock Protein 70 kDa Heat Shock Protein peptideaa 106-125; aa 1-20; aa 121-140; aa 136-155; aa 151-170; aa 16-35; aa166-185; aa 181-199; aa 195-214; aa 210-229; aa 225-244; aa 240- 259; aa255-275; aa 271-290; aa 286-305; aa 301-320; aa 31-50; aa 316-335; aa331-350; aa 346-365; aa 361-380; aa 376-395; aa 391-410; aa 406-425; aa421-440; aa 436- 455; aa 451-470; aa 466-485; aa 46-65; aa 481-500; aa496-515; aa 511-530; aa 526-545; aa 541-560; aa 556- 575; aa 571-590; aa586-605; aa 601-620; aa 616-635; aa 61-80; aa 631-640; aa 76-95; or aa91-110 71 kDa Heat Shock Protein M. tuberculosis 90 kDa Heat ShockProtein GroEL CNS 2′,3′-cyclic nucleotide 3′-phosphodiesterase peptideaa 106- 125; aa 1-20; aa 121-140; aa 136-155; aa 151-170; aa 16-35; aa166-185; aa 181-200; aa 196-215; aa 211-230; aa 226- 245; aa 241-260; aa256-275; aa 271-290; aa 286-305; aa 301-320; aa 31-50; aa 316-335; aa331-350; aa 346-365; aa 361-380; aa 376-395; aa 391-410; aa 406-421; aa46-65; aa 61-80; aa 76-95; or aa 91-110 Acetyl Cholinesterase ADAM-10alpha-Cristallin beta-Cristallin bovine Myelin Basic Protein BrainExtract I Brain Extract II Brain Extract III Glial Filament AcidicProtein Research Diagnostic guinea pig Myelin Basic Protein human MyelinBasic Protein Myelin-Associated Oligodendrocytic Basic Protein peptideaa 106-125; aa 1-20; aa 121-140; aa 136-155; aa 151-170; aa 16-35; aa166-185; aa 181-200; aa 31-50; aa 46-65; aa 61-80; aa 76-95; aa 91-110;aa 106-125; aa 1-20; aa 121- 140; aa 136-155; or aa 151-170Myelin/oligodendrocyte glycoprotein peptide aa 16-35; aa 166-185; aa181-200; aa 196-215; aa 211-230; aa 226-247; aa 31-50; aa 35-55; aa46-65; aa 61-80; aa 76-95; or aa 91- 110 murine Myelin Basic ProteinMyelin Associated Glycoprotein Myelin Basic Protein peptide aa 104-123;aa 11-30; aa 113- 132; aa 1-20; aa 121-138; aa 124-142; aa 138-147; aa141- 161; aa 143-168; aa 155-178; aa 26-35; aa 31-50; aa 41-60; aa51-70; aa 61-80; aa 71-92; aa 84-94; aa 89-101; aa 173- 186; or aa93-112 Myelin Protein 2 peptide aa 106-125; aa 1-20; aa 121-132; aa16-35; aa 31-50; aa 46-65; aa 61-80; aa 76-95; or aa 91- 110Neurofilament 160 kd Neurofilament 200 kd Neurofilament 68 kd NeuronalEnolase Nicastrin NMDA receptor NOGO Olygodendrocyte-Specific Proteinpeptide aa 106-125; aa 1-20; aa 121-140; aa 136-155; aa 151-170; aa16-35; aa 166-185; aa 181-199; aa 195-217; aa 31-50; aa 46-65; aa 61-80;aa 76-95; or aa 91-110 Proteolipid Protein Proteolipid Protein peptideaa 100-119; aa 10-29; aa 110- 129; aa 1-19; aa 125-141; aa 137-150; aa137-154; aa 150- 163; aa 151-173; aa 158-166; aa 161-180; aa 178-191; aa180-199; aa 190-209; aa 20-39; aa 205-220; aa 215-232; aa 220-239; aa220-249; aa 248-259; aa 250-269; aa 265-277; aa 35-50; aa 40-59; aa50-69; aa 65-84; aa 80-99; or aa 91- 110 Retinol Binding ProteinS100beta protein Assay Designs Super Oxide Dismutase Synuclein, betaSynuclein, gamma Tissue Amydgala Amydgala AD Brain lysate Brain TissueMembrane Cerebellar pedunculus Cerebral meninges Corpus Callosum CorpusCallosum AD Diencephalon Fetal brain Frontal lobe Frontal lobe ADHippocampus Hippocampus AD Insula Occipital lobe Occipital lobe ADOlfactory region Optic Nerve Parietal lobe Parietal lobe AD Pons Pons ADPostcentral gyrus Postcentral gyrus AD Precentral gyrus Precentral gyrusAD Spinal cord Temporal lobe Temporal lobe AD Thalamus Thalamus ADAmyloid beta AD related Amyloid beta 10-20 Amyloid beta peptide 1-12;12-28; 1-23; 1-38; beta 17-40; 25-35; or 34-42 Amyloid bri proteinprecursor 227 Amyloid DAN Protein Fragment 1-34 Amyloid PrecursorProtein Amyloid protein no AB component Secreted amyloid precursorprotein (SAP) beta Tau isoform variant 0N3R Tau isoform variant 1N3R Tauisoform variant 0N4R Tau isoform variant 2N3R Tau phospho Ser412 Tauphospho Ser441 Tau phospho Thr181 Tau Protein human Lipids (±)9-HODECayman Chemical 1 Palmitoyl-2-(5′oxo-Valeroyl)-sn-Glycero-3-Phosphocholine 15a-hydroxycholestene 15-ketocholestane 15-ketocholestene1-Palmitoil-2-(9′oxo-Nonanoyl)-sn-Glycero-3- Phosphocholine1-Palmitoil-2-Azelaoyl-sn-Glycero-3-Phosphocholine1-Palmitoil-2-Glutaroyl-sn-Glycero-3-Phosphocholine 5 α-cholestane-3 β,15 α-diol 9(S)-HODE Cayman Chemical Asialoganglioside-GM1Asialoganglioside-GM2 Brain ceramides Brain D-erythrosphingosine Brainlysophosphatidylethanolamine Brain L-α-lysophosphatidylserine BrainL-α-phosphatidylcholine Brain L-α-phosphatidyl-ethanolamine BrainL-α-phosphatidylserine Brain polar lipid extract Brain sphingomyelinBrain sulfatide Brain total lipid extract Cardiolipin Ceramide Ceramide1-phosphate Cholesterol Disialogaglioside-GD1B Disialogaglioside-GD2Disialoganglio side GD1a Disialoganglioside GD3 Fucosyl-GM1Galactocerebrosides Ganglioside Mixture Ganglioside-GM4Gangliotetraosylceramide asialo-GM1 HDL Hexacosanoic acid (26) Hydroxyfatty acid ceramide Isoprostane F2 I Lactocerebrosides LactosylceramideLDL Lipid A, diphosphoryl from Salmonella enterica Lipopolysaccharidesfrom Escherichia coli Lipopolysaccharides from Pseudomona aeruginosaLipopolysaccharides from Salmonella enterica Lyso-GM1Monosialoganglioside GM1 Monosialoganglioside GM2 MonosialogangliosideGM3 N-Hexanoyl-D-sphingosin Non-hydroxy fatty acid ceramidePhosphatidylinositol-4 phosphate Squalene Sulfatides Tetracosanoic acid(24) Tetrasialoganglioside-GQ1B TNPAL Galactocerebroside Total braingangliosides Total cerebroside Trisialoganglioside GT1aTrisialoganglioside-GT1B

The control of EAE by AHR activation correlated with a significantdecrease in IgG serum antibodies to 97 myelin antigens, which are listedin Table 2.

TABLE 2 Specificity of IgG Antibodies Showing a Significant (FDR < 0.05)Downregulation in TCDD-Treated Mice Antigen FDR 70 kDa. Heat ShockProtein peptide aa 331-350 1.78E−05 60 kDa. Heat Shock Protein peptideaa 255-275 0.00547 60 kDa. Heat Shock Protein peptide aa 13-35 0.0054732 kDa. Heat Shock protein 0.00547 Myelin Basic Protein peptide aa138-147 0.00547 Proteolipid Protein peptide aa 1-19 0.00547 ProteolipidProtein peptide aa 161-180 0.00547 Proteolipid Protein peptide aa 10-290.00547 60 kDa. Heat Shock Protein peptide aa 1-20 0.0055 70 kDa. HeatShock Protein peptide aa 61-80 0.0055 Ceramide 0.0055 Myelin-AssociatedOligodendrocytic Basic Protein peptide aa 91-110 0.0055 ProteolipidProtein peptide aa 137-150 0.0055 NOGO 0.00557 Olygodendrocyte-SpecificProtein peptide aa 76-95 0.00557 b-Cristallin 0.0058 Myelin-AssociatedOligodendrocytic Basic Protein peptide aa 121-140 0.00703 60 kDa. HeatShock Protein peptide aa 225-244 0.00708 Myelin Basic Protein peptide aa113-132 0.00925 Olygodendrocyte-Specific Protein peptide aa 46-650.00925 Myelin Protein 2 peptide aa 91-110 0.00925 Myelin-AssociatedOligodendrocytic Basic Protein peptide aa 151-170 0.0093Myelin/oligodendrocyte glycoprotein peptide aa 31-50 0.0093 NT-3 0.0093Proteolipid Protein peptide aa 40-59 0.0116 70 kDa. Heat Shock Proteinpeptide aa 421-440 0.0118 Myelin Basic Protein peptide aa 173-186 0.012570 kDa. Heat Shock Protein peptide aa 121-140 0.0132 2′,3′-cyclicnucleotide 3′-phosphodiesterase peptide aa 391-410 0.0132Olygodendrocyte-Specific Protein peptide aa 136-155 0.0132Olygodendrocyte-Specific Protein peptide aa 106-125 0.0134 70 kDa. HeatShock Protein peptide aa 136-155 0.0141 2′,3′-cyclic nucleotide3′-phosphodiesterase peptide aa 406-421 0.0141 Myelin-AssociatedOligodendrocytic Basic Protein peptide aa 166-185 0.0143 Myelin Protein2 peptide aa 1-20 0.0143 Myelin Protein 2 peptide aa 76-95 0.0144Proteolipid Protein peptide aa 125-141 0.0144 Proteolipid Proteinpeptide aa 178-191 0.0144 40 kDa. Heat Shock Protein 0.0145 2′,3′-cyclicnucleotide 3′-phosphodiesterase peptide aa 106-125 0.0158Olygodendrocyte-Specific Protein peptide aa 195-217 0.0174 2′,3′-cyclicnucleotide 3′-phosphodiesterase peptide aa 240-259 0.0187 70 kDa. HeatShock Protein peptide aa 76-95 0.0194 Proteolipid Protein peptide aa265-277 0.0194 Myelin Basic Protein peptide aa 89-101 0.0199 MyelinBasic Protein peptide aa 71-92 0.0199 Myelin-Associated OligodendrocyticBasic Protein peptide aa 16-35 0.0199 Proteolipid Protein peptide aa265-277 0.0199 60 kDa. Heat Shock Protein peptide aa 46-65 0.0241 70kDa. Heat Shock Protein peptide aa 166-185 0.0241 2′,3′-cyclicnucleotide 3′-phosphodiesterase peptide aa 151-170 0.0241 2′,3′-cyclicnucleotide 3′-phosphodiesterase peptide aa 376-395 0.0241 Myelin BasicProtein peptide aa 11-30 0.0241 Myelin/oligodendrocyte glycoproteinpeptide aa 211-230 0.0241 Proteolipid Protein peptide aa 265-277 0.024170 kDa. Heat Shock Protein peptide aa 181-199 0.0242Olygodendrocyte-Specific Protein peptide aa 31-50 0.0242 ProteolipidProtein peptide aa 265-277 0.0242 Myelin/oligodendrocyte glycoproteinpeptide aa 91-110 0.0249 Optic Nerve lysate 0.0249 2′,3′-cyclicnucleotide 3′-phosphodiesterase peptide aa 361-380 0.0258Lactosylceramide 0.0258 Myelin Protein 2 peptide aa 31-50 0.0258 MyelinBasic Protein peptide aa 1-20 0.028 NMDA receptor 0.0285 CNF 0.02892′,3′-cyclic nucleotide 3′-phosphodiesterase peptide aa 136-155 0.0292Myelin Basic Protein peptide aa 141-161 0.0298 70 kDa. Heat ShockProtein peptide aa 406-425 0.0307 2′,3′-cyclic nucleotide3′-phosphodiesterase peptide aa 210-229 0.0307 Galactocerebrosides0.0307 Myelin/oligodendrocyte glycoprotein peptide aa 46-65 0.0307Proteolipid Protein peptide aa 150-163 0.0307 Proteolipid Proteinpeptide aa 265-277 0.0307 Proteolipid Protein peptide aa 80-99 0.0307 60kDa. Heat Shock Protein peptide aa 210-229 0.0323 Proteolipid Proteinpeptide aa 137-154 0.0324 2′,3′-cyclic nucleotide 3′-phosphodiesterasepeptide aa 1-20 0.0337 2′,3′-cyclic nucleotide 3′-phosphodiesterasepeptide aa 225-244 0.0337 Myelin-Associated Oligodendrocytic BasicProtein peptide aa 61-80 0.0337 Proteolipid Protein peptide aa 158-1660.0337 Ceramide 1 phosphate 0.0346 Myelin-Associated OligodendrocyticBasic Protein peptide aa 136-155 0.0369 Myelin Basic Protein peptide aa155-178 0.0379 Myelin/oligodendrocyte glycoprotein peptide aa 106-1250.0392 Proteolipid Protein peptide aa 180-199 0.0408 Myelin Protein 2peptide aa 121-132 0.0413 Myelin Basic Protein peptide aa 104-123 0.041970 kDa. Heat Shock Protein 0.0421 Non h fatty acid ceramide 0.0421Myelin-Associated Glycoprotein 0.0452 Myelin Basic Protein peptide aa143-168 0.0452 2′,3′-cyclic nucleotide 3′-phosphodiesterase peptide aa91-110 0.047 2′,3′-cyclic nucleotide 3′-phosphodiesterase peptide aa181-199 0.0476 70 kDa. Heat Shock Protein peptide aa 255-275 0.0486Brain ceramides 0.0486 Myelin Protein 2 peptide aa 46-65 0.0496

To further characterize the suppression of EAE by AHR activation westudied the activity of myelin specific T cells induced by vaccinationwith MOG₃₅₋₅₅/CFA in TCDD-treated mice. TCDD-treated mice showed asuppressed recall proliferative response to the MOG₃₅₋₅₅ peptide,however no differences were seen upon activation with antibodies to CD3(see FIGS. 4c-d ).

In addition, cells were stimulated in culture medium containing 100μg/ml MOG₃₅₋₅₅ for 2 days or with PMA (50 ng/ml) (Sigma-Aldrich) andionomycin (1 nM) (Calbiochem, San Diego, Calif., USA) for 4 hours,Golgistop (BD Biosciences) was added to the culture during the last 4hours. After staining of surface markers, cells were fixed andpermeabilized using Cytofix/Cytoperm and Perm/Wash buffer from BDBiosciences according to the manufacturer's instructions. All antibodiesto cytokines (IFN-gamma, IL-17, IL-10) including the correspondingisotype controls were obtained from BD Biosciences. Cells were incubated(1:100) at 25° C. for 20 min and washed twice in Perm/Wash beforeanalysis. Data were acquired on a FACSCalibur (BD Biosciences) andanalyzed with FlowJo software (Tree Star, Ashland, Oreg., USA). Whencompared to the draining lymph node cells from control animals, cellsfrom TCDD-treated mice secreted higher amounts of TGFb1 and loweramounts of IFNg and IL-17 upon activation with MOG₃₅-5₅ (see FIG. 4e );we did not detect significant amounts of IL-4 or IL-10. Moreover, AHRactivation with TCDD led to a decrease in the frequency of CD4+IL-17 andCD4⁺IFNg⁺ T cells in the draining lymph nodes (see FIG. 4F).

These data suggest that AHR activation interferes with the generation ofthe encephalitogenic T cell response.

Example 6: Treg Induced by AHR Activation Suppress EAE by aTGFb1-Dependent Mechanism

The inhibition of the development of EAE by AHR activation with TCDD wasassociated with a significant increase in the frequency of CD4⁺Foxp3⁺ Tcells (see FIG. 5A). To identify the mechanism responsible for thedecreased proliferation to MOG₃₅₋₅₅ in TCDD-treated animals shown inFIG. 4D, the CD4⁺CD25⁺ Treg population was depleted with magnetic beads.Treg depletion recovered the recall response to MOG₃₅₋₅₅ in immunizedmice treated with TCDD (see FIG. 5B), suggesting that the suppressionobserved in FIG. 4D resulted from the activity of the TCDD-induced Treg(see FIG. 5A). Moreover, protection from EAE could be transferred towild type naïve animals by the transfer of 5×10⁶ CD4⁺ T cells fromTCDD-treated mice, but not with cells isolated from vehicle-treated mice(P<0.001, two-way ANOVA, n=4; FIG. 5C). The control of the pathogenic Tcell response was mediated by CD4⁺CD25⁺ Treg, their depletion abrogatedthe protective effect of the transferred cells (P<0.001, two-way ANOVA,n=4; FIG. 5C).

Further characterization revealed that effector CD4⁺Foxp3:GFP⁻ T cellspurified from TCDD-treated mice showed normal proliferation (FIG. 5F),but significantly decreased secretion of IL-17 and IFNγ upon activationwith MOG₃₅₋₅₅ (see FIG. 5G).

To confirm that the protective effect of TCDD on EAE was Treg mediatedwe depleted the natural Treg with antibodies to CD25 prior to TCDDtreatment. The difference between undepleted and depleted cellpopulations are shown in FIG. 5H. TCDD-treated mice showed a fasterrebound in their Treg numbers (P<0.04 at day 7) (see FIG. 5H),concomitant with a significant delay in the onset of EAE (P<0.03) and asignificant reduction in IL-17+CD4+ T cells in the draining lymph nodes(P<0.03; see FIGS. 5I and 5J). Moreover, the transfer of 5×10⁶ CD4⁺ Tcells from TCDD-treated mice significantly inhibited the development ofEAE, as shown in FIG. 5h and Table 5. This protective effect was lostwhen CD4⁺CD25⁺ T cells were depleted, see FIG. 5h and Table 5. Together,these data suggest that AHR activation by TCDD results in the generationof CD4+ Foxp3+ Treg that control the encephalitogenic response.

TABLE 5 EAE Suppression in Treg Depleted Cell Populations Incidence Meanday of onset Mean maximum Treatment (positive/total) (Mean ± SD) score(mean ± SD) CD4⁺ control  7/7 (100%) 12.3 ± 1.9 2.7 ± 1.0 CD4⁺ TCDD 3/6(57%) 13.3 ± 0.6  0.7 ± 0.8* CD4⁺CD25⁻ 3/4 (75%) 11.0 ± 0.0 2.6 ± 1.8TCDD Naïve C57BL/6 mice received CD4⁺ or CD4⁺CD25⁻ T cells (5 × 10⁶)purified from TCDD or control treated mice 10 days after immunizationwith MOG₃₅₋₅₅/CFA. 24 hours later EAE was induced in the recipient micewith MOG₃₅₋₅₅/CFA, and the mice were monitored for EAE development.Statistical analysis was performed by comparing groups using one-wayanalysis of variance. *P < 0.05 vs CD4⁺ control group.

TGFb1 has been linked to the suppressive activity of Treg in vitro andin vivo (Li et al., Annu Rev Immunol. 24, 99-146 (2006)). To asses therole played by TGFb1 in the inhibition of the recall response toMOG₃₅₋₅₅ by Treg (see FIGS. 4d and 5b ), we activated lymph node cellsfrom TCDD treated mice in the presence of blocking antibodies to IL-4,IL-10, TGFb1, or an isotype-matched control. FIG. 5d shows thatincubation with antibodies to TGFb1, but not to IL-4 or IL-10 couldrecover the recall response to MOG₃₅₋₅₅.

To analyze the role played by TFGb1 in vivo in the control of EAE, wetransferred CD4⁺ T cells from TCDD treated mice into naïve miceexpressing a dominant negative variant of the TGFb receptor II on theirT cells; T cells from these mice are unresponsive to theimmunosuppressive effects of TGFb1 (Gorelik et al., Immunity. 12, 171-81(2000)). As shown in FIG. 5E, transferred Treg cells could control EAEin wild type mice but not in mice harboring T cells unresponsive toTGFb1 (P<0.001, two-way ANOVA, n=4). Thus, Treg induced by theactivation of AHR with TCDD inhibit the progression of EAE by aTGFb1-dependent mechanism.

Example 7: Endogenous AHR Ligands Control Treg Development In Vivo

The observations described herein regarding the control of Tregdevelopment by AHR activation suggest that endogenous AHR ligandsparticipate in immune regulation. In support of this, we havedemonstrated that naïve AHR-d mice harbor lower levels of CD4⁺Foxp3⁺ Tcells (P<0.03, t-test; see FIG. 6A), and higher levels ofCD4⁺CD25⁺Foxp3⁻ T cells. In addition, we have demonstrated that thesecells develop a significantly stronger EAE, which is characterized by anearlier disease onset and a higher clinical score (P<0.001, two-wayANOVA, n=6-8; see FIG. 6B).

Several endogenous AHR ligands are described in the art²⁸. Based on ourresults, AHR ligands such as TCDD could be useful in the control of Tregdevelopment. Our data additionally demonstrate that AHR ligands such asTCDD can be used to suppress the development and/or progression of EAE.Clearly, such technology would also be useful in the modulation of otherimmunological disorders such as autoimmune disorders. Two additionalendogenous high affinity ligands for AHR are tryptamine (TA)(Heath-Pagliuso et al., Biochemistry. 37, 11508-15 (1998)), a derivativeof tryptophan (Trp) catabolism and2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE)(Song et al., Proc Natl Acad Sci USA. 99, 14694-9 Epub 2002 Oct. 30(2002)), a molecule isolated from the lung. Interestingly, no toxicityhas been reported for these AHR ligands in vivo, probably as a result oftheir short half-life (Henry et al., Arch Biochem Biophys. 450, 67-77Epub 2006 Mar. 3 (2006)). To confirm the physiologic relevance of AHRactivation for the control of Treg activity, we tested the effect of TAand ITE on EAE. Based on the short half-life of these molecules weadministered them on a daily basis. The administration of ITE, but notTA, led to a significant reduction on EAE severity (P<0.001, two-wayANOVA, n=9; see FIG. 6c ), likely due to rapid degradation of TA. Thisobservation suggests that endogenous AHR ligands participate in thecontrol of inflammation under physiological conditions.

Together, these results indicate that modulation of Foxp3 expression bymodulating activity of a transcription factor that binds to Foxp3 can beused to affect Treg and control of the immune response in vivo.

Example 8: Expression Levels of Transcription Factors in Foxp3 Knock-inMice

Mouse Treg and non-Treg were isolated from Foxp3gpf knock in mice, mRNAwas prepared and Foxp3 (see FIG. 8A), NKX2.2 (see FIG. 8B), EGR1 (seeFIG. 8C), EGR2 (see FIG. 8D) and EGR3 (see FIG. 8E) expression wasquantified by real time PCR. Foxp3gpf knock in mice have a GFP reporterinserted in the Foxp3 gene, producing GFP in Foxp3⁺ Treg and thereforefacilitating the identification and FACS sorting of GFP:Foxp3⁺ Treg.

GFP⁻ CD4⁺ T cells were isolated from Foxp3gpf knock in mice, and thenwere activated in vitro with antibodies to CD3 and CD28 in the presenceof TGFβ1 to induce Treg differentiation in vitro. mRNA was prepared atthe beginning of the experiment and after 3 or 6 days in culture, andthe expression of Foxp3 (see FIG. 9A), NKX2.2 (see FIG. 9B), EGR1 (seeFIG. 9C), EGR2 (see FIG. 9D) and EGR3 (see FIG. 9E) expression wasquantified in the Foxp3:GFP⁺CD4⁺ T cells by real time PCR.

These results indicate that expression of Foxp3 and the transcriptionfactors EGR1, EGR2, EGR3 and NKX2.2 is correlated. Taken together, thereported effects that these TFs exert on the regulation of geneexpression, the identification of TF binding sites on the Foxp3 gene,and the correlation between the expression of these TF and Fox3, suggestthat EGR1, EGR2, EGR3 and NKX2.2 play a role in the regulation of Foxp3expression and the generation of Treg.

Example 9: Combination Treatment Using Tryptamine (TA)

As shown above, TA is rapidly degraded in vivo by monoamine oxidaseinhibitors. As shown in FIG. 13, when combined with the monoamineoxidase inhibitor trans-2-Phenylcyclopropylamine hydrochloride(Tranylcypromine), TA effectively suppresses EAE suppression.

This observation suggests that TA is a TCDD-like ligand that, when usedin combination with a monoamine oxidase inhibitor, can be used as atranscription factor ligand for promoting an increase in the numberand/or activity of Treg.

Example 10: Modified Screening Assays

A modified zebrafish based screening assay was established bymicroinjecting fertilized zebrafish eggs with a BAC construct encodingthe complete mouse Foxp3 locus, with a renilla reporter inserted afterthe Foxp3 methionine start codon (ATG). Six days after microinjection,renilla activity was determined in total zebrafish lysates. As shown inFIG. 14, murine Foxp3 was expressed in the microinjected fish asdetermined by renilla luciferase activity. The activity increased in thepresence of TCDD in a dose-dependent manner.

These data suggest that zebrafish lines encoding murine Foxp3 can beused to screen for small molecules that increase or decrease Foxp3expression levels.

Example 11: AHR Activation with its Non-Toxic Ligand ITE InducesFunctional Treg

The ligand-activated transcription factor aryl hydrocarbon receptor(AHR) is a regulator of zebrafish, mouse and human Foxp3 expression andT_(reg) differentiation (Quintana et al., Nature 23, 23 (2008)). AHRactivation by its ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)induced T_(reg) that suppressed experimental autoimmuneencephalomyelitis (EAE) by a TGFb1-dependent mechanism. These findingsidentify AHR as a therapeutic target of interest for the management ofautoimmune disorders, but its therapeutic exploitation is limited by thewell-characterized toxic features of TCDD (Baccarelli et al., EnvironHealth Perspect. 110, 1169 (2002)).

Several endogenous AHR ligands have been isolated, among them tryptophanderivatives like tryptamine (TA) (Heath-Pagliuso et al., Biochemistry.37, 11508 (1998)) and the mucosal associated2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE)depicted in FIG. 1 (Song et al., Proc Natl Acad Sci USA. 99, 14694 (Nov.12, 2002)). Notably, although ITE and TA have been shown to be highaffinity AHR ligands, they do not display the toxic effects reported byTCDD (Heath-Pagliuso et al., (1998), supra; Henry et al., Arch BiochemBiophys. 450, 67 (2006)). As demonstrated herein, the non-toxic AHRligand ITE administered intraperitoneally, orally or with pegylated goldnanoparticles can be used to induce functional T_(reg).

To analyze the feasibility of using ITE to activate AHR in vivo in atherapeutic setup, we studied the effect of ITE on EAE development. EAEwas induced on naïve C57BL/6 mice and ITE (200 mg/mice) was administeredorally or intraperitoneally on daily basis. ITE administration, eitherorally or intraperitoneally, resulted in a significant delay on EAEdevelopment and a significant reduction of EAE clinical score (FIGS.15A-B). Thus, AHR activation by ITE induces functional T_(reg) that cancontrol EAE.

To study the mechanism by which the ITE-induced T_(reg) control EAE westudied the ability of AHR activation by ITE to induce T_(reg). Wetreated naïve C57BL/6 mice with ITE (200 mg/mouse administered ip,daily) and immunized them with 100 mg/mouse of MOG₃₅₋₅₅ in CFA. Spleenswere prepared 10 days later and CD4⁺FoxP3⁺ T_(reg) were quantified byFACS. Administration of ITE led to a significant increase in the numberof CD4⁺FoxP3⁺ T_(reg) (FIGS. 16A-B and 17A-B). Notably, this increaseresulted from the expansion of both CD25⁺ and CD25-CD4⁺FoxP3⁺ T_(reg),but did lead to significant alterations in the levels of LAP⁺ regulatoryT cells (FIGS. 16A-B and 17A-B). Thus AHR activation by ITE results inthe expansion of the CD4⁺FoxP3⁺ T_(reg) compartment.

To confirm the lack of toxicity of ITE, we administered itintraperitoneally for 14 days, 200 mg/mouse and studied the blood levelsof biochemical indicators of liver function induction. Hepatocites areknown to express high levels of AHR, thus toxic effects of AHRactivation are manifested in the liver. Table 6 shows that at day 14 wedid not detect any significant difference in the biochemical indicatorsof liver function, confirming the lack of toxicity in ITE.

TABLE 6 ITE Administration Does Not Result in Liver Toxicity TEST UnitsControl ITE Reference Range ALT/GPT U/L 16 ± 2  19 ± 4   0-54 AST/GOTU/L 79 ± 29 94 ± 17  9-74 Alkaline Phosphatase U/L 75 ± 8  55 ± 11 36-300 Total Bilirubin mg/dL 0 ± 0 0 ± 0 0.1-1.2 Direct Bilirubin mg/dL0 ± 0 0 ± 0 0.0-0.8 Total Protein g/dL 6 ± 0 5 ± 0 4.4-8.0 Albumin g/dL2 ± 0 3 ± 0 2.9-5.4 Globulin g/dL 3 ± 0 3 ± 0 2.0-4.0

To study the feasibility of administering ITE orally to activate AHR andinduce functional Treg we treated naïve C57BL/6 mice with ITE (200mg/mouse administered orally, daily) and immunized them with 100mg/mouse of MOG₃₅₋₅₅ in CFA. Spleens were prepared 10 days later andCD4⁺FoxP3⁺ T_(reg) were quantified by FACS. Administration of ITE led toa significant increase in the number of CD4⁺FoxP3⁺ T_(reg) (FIGS. 18A-Band 19A-B). Notably, this increase resulted from the expansion of bothCD25⁺ and CD25⁻ CD4⁺FoxP3⁺ T_(reg), but did lead to significantalterations in the levels of LAP⁺ regulatory T cells (FIGS. 18A-B and19A-B). Thus ITE can be administered orally to activate AHR and expandthe CD4⁺FoxP3⁺ T_(reg) compartment.

To confirm the ability of AHR activation by ITE to expand the T_(reg)compartment we used Foxp3^(gpf) knock in mice. Foxp3^(gpf) knock in micehave a GFP reporter inserted in the Foxp3 gene, producing a GFP:Foxp3fusion protein that facilitates the identification and FACS sorting ofGFP:FoxP3⁺ T_(reg) (Bettelli et al., Nature 441, 235 (2006)).Foxp3^(gpf) knock in mice were treated with ITE (200 mg/mouseadministered ip, daily) and immunized with 100 mg/mouse of MOG₃₅₋₅₅ inCFA. Ten days later and CD4⁺FoxP3:GFP⁺ T_(reg) were quantified by FACS.Administration of ITE led to a significant increase in the number ofCD4⁺FoxP3:GFP⁺ T_(reg) in blood and spleen (FIGS. 20A-B and 21A-B). ThusAHR activation by ITE results in the expansion of the CD4⁺FoxP3⁺ T_(reg)compartment.

We then studied the effect of ITE administration on the encephalitogenicresponse against myelin. EAE was induced on naïve C57BL/6 mice and ITE(200 mg/mice) was administered orally or intraperitoneally on dailybasis. Ten days after vaccination with MOG₃₅₋₅₅/CFA, ITE-treated miceshowed a suppressed recall proliferative response to the MOG₃₅₋₅₅peptide (FIG. 22); no differences were seen upon activation withantibodies to CD3 (FIG. 22). When compared to the splenocytes fromcontrol animals, CD4⁺ T cells from ITE-treated mice secreted higheramounts of TGFb1 and IL-10 and lower amounts of IL2, IL6, IFNg and IL17upon activation with MOG₃₅₋₅₅ (FIG. 22). Similar results were observedon the recall response to MOG₃₅₋₅₅ of mice treated with orallyadministered ITE (FIG. 23).

To confirm the suppressive effects of AHR activation on the generationof T cells secreting IFNg and IL17, IFNg⁺ and IL17⁺CD4⁺ T cells werequantified by FACS in the draining lymph nodes ten days after footpadimmunization and intraperitoneal administration of ITE (200 mg/mice).AHR activation with ITE led to a decrease in the frequency of CD4+IL17⁺and CD4⁺IFNg⁺ T cells (FIG. 24). In accordance with these results, wefound a significant reduction in the secretion of IL-17 and IFNg bylymph node cells from ITE-treated mice activated with MOG₃₅₋₅₅ or αCD3.

To investigate the effect of AHR activation by ITE on the frequency ofMOG₃₅₋₅₅ specific Treg and effector T cells (Teff), Foxp3^(gpf) knock inmice were immunized with MOG₃₅₋₅₅/CFA, treated daily withintreaperitoneal ITE (200 mg/mice) and MOG₃₅₋₅₅-specific T_(reg)(CD4⁺FoxP3:GFP⁺) and Teff (CD4⁺FoxP3:GFP⁻) were analyzed by FACS usingrecombinant MHC class II tetramers containing MBP₃₅₋₅₅ or the controlpeptide TMEV₇₀₋₈₆. AHR activation with ITE led to a decrease in thefrequency of MOG₃₅₋₅₅-specific Teff and to a concomitant increase in thefrequency of MOG₃₅₋₅₅-specific T_(reg), reducing the MOG₃₅₋₅₅-specificTeff/T_(reg) ratio by half (FIG. 25).

To investigate the active suppression of MOG₃₅₋₅₅-specific Teff by Treg,Foxp3^(gpf) knock in mice were immunized with MOG₃₅₋₅₅/CFA, treateddaily with intreaperitoneal ITE (200 mg/mice) and the recall response toMOG₃₅₋₅₅ and a mitogenic antibody to CD3 antibody was studied onFACS-sorted CD4⁺ T cells and CD4⁺ FoxP3:GFP− Teff. Purified CD4+ T cellsfrom ITE-treated mice showed a suppressed response to MOG₃₅₋₅₅ but notto anti-CD3 (FIG. 26). This suppressed response to MOG₃₅₋₅₅ was lostupon removal of the CD4⁺FoxP3:GFP⁺ T_(reg) (FIG. 26). To further analyzethe MOG₃₅₋₅₅-specific suppressive activity of the CD4⁺FoxP3:GFP⁺T_(reg), they were cocultured at different ratios and assayed for thesuppression of MOG₃₅₋₅₅ or anti-CD3-triggered proliferation ofCD4⁺FoxP3:GFP− Teff form 2D2 mice, which harbor a TCR specific forMOG₃₅₋₅₅. CD4⁺FoxP3:GFP⁺ T_(reg), from ITE-treated mice displayed anincreased MOG₃₅₋₅₅-specific suppressive activity, which could beinhibited with antibodies blocking antibodies to TGFb1 (FIGS. 27A-C).All in all, these data suggests that, similarly to what we havedescribed for TCDD, AHR activation by ITE results in the expansion ofantigen-specific CD4⁺ FoxP3⁺ T_(reg) that suppress the encephalitogenicresponse in a TGFb1-dependent manner.

To demonstrate that the effect of ITE on EAE was mediated by T_(reg), wepurified CD4⁺ T cells from mice protected from EAE by oral orintraperitoneal administration of ITE 14 days after EAE induction.Protection from EAE could be transferred to wild type naïve animals bythe transfer of 5 10⁶ CD4⁺ T cells ITE-treated mice, but not with cellsisolated from vehicle-treated mice (FIG. 28). The control of thepathogenic T cell response was mediated by CD4⁺CD25⁺ T_(reg), theirdepletion from the transferred population abrogated the protectiveeffect of the transferred cells. Thus, the T_(reg) induced by theactivation of AHR with ITE inhibit the progression of EAE.

AHR is known to be expressed by antigen presenting cells (APC) such asdendritic cells (CD11c⁺) and macrophages (CD11b⁺) (Vorderstrasse andKerkvliet, Toxicol Appl Pharmacol. 171, 117 (2001); Laupeze et al., JImmunol. 168, 2652 (2002); Hayashi et al., Carcinogenesis. 16, 1403(1995); Komura et al., Mol Cell Biochem. 226, 107 (2001)). To analyzethe effects that AHR activation by ITE might have on different APCpopulations, which can potentially influence the generation of Teff andT_(reg) cells, we studied the effect of ITE and TCDD treatment on MHCclass II expression by as dendritic cells (CD11c⁺) and macrophages(CD11b). C57BL/6 mice were treated with ITE (200 mg/mouse administeredip, daily) or TCDD (1 mg/mouse administered ip on day 0) and immunizedthem with 100 mg/mouse of MOG₃₅₋₅₅ in CFA. Spleens were prepared 10 dayslater and MCH class I expression was investigated on CD11b⁺ and CD11c⁺cells by FACS. Administration of ITE or TCDD resulted in a significantdecrease in CD11c⁺ MHC class II expression, which was concomitant with asignificant increase in CD11b⁺ MHC class II expression (FIG. 29). SinceCD11c⁺ MHC-II⁺ and CD11 b⁺ MHC-II⁺ have been recently linked to theinduction of Teff and T_(reg), respectively, these results suggest thatchanges in the different APC populations might contribute to theimmunomodulatory effects of AHR activation by ITE.

Example 12: Administration of ITE-Loaded Nanoparticles InducesFunctional Treg

As noted above, administration of a single dose of 1 mg/mouse of the AHRligand TCDD could prevent the development of EAE. To achieve similareffects on disease progression, 200 mg/mouse of ITE have to beadministered daily throughout the experiment. ITE is a tryptophanderivative which is thought to have a short half-life in vivo as aresult of the activity of specific enzymes. Indeed, administration ofITE at weekly intervals, instead of daily, results in a complete loss oftis protective effects on EAE (FIG. 30).

Gold colloid has been in use for over 50 years in the treatment ofrheumatoid arthritis, these gold colloid nanoparticles have been shownto have little to no long-term toxicity or adverse effects (Paciotti etal., Drug Deliv. 11, 169 (2004)). Due to their small size (10-100 nmdiameter), gold colloid nanoparticles have large surface areas on whichmultiple small proteins or other molecules can be conjugated (Paciottiet al., Drug Deliv. 11, 169 (2004)). The PEGylation of gold colloidnanoparticles greatly enhances the overall stability of the molecule towhich it is covalently bonded (Qian et al., Nat Biotechnol. 26, 83(2008)). Moreover, recently it has been shown that PEGylated) goldcolloid nanoparticles can be linked to specific antibodies to targetthem to specific cell types (Qian et al., Nat Biotechnol. 26, 83(2008)). Thus, to increase the half-life of ITE and to facilitate itstargeting to specific cell types, we constructed polyethylene glycolcoated (PEGylated) gold colloid nanoparticles loaded with AHR ligands(FIG. 31).

PEGylated gold colloid nanoparticles carrying the AHR ligands FICZ, ITEor TCDD showed a typical spectrum of optical absorption (FIG. 32).Moreover, FICZ, ITE or TCDD-loaded nanoparticles activated luciferaseexpression on an AHR-reporter cell line to levels similar to thoseachieved by 10 nM TCDD.

To investigate the in vivo functionality of AHR-ligand loadednanoparticles we induced EAE on naïve C57BL/6 mice and treated them,starting at day 0, weekly with 45 femtomoles of nanoparticles. Similarlyto what we have described in our previous experiments, treatment withTCDD resulted in a complete suppression of EAE, while the AHR ligandFICZ worsened the disease (FIG. 33). Weekly administration of ITE-loadednanoparticles resulted in a significant inhibition of EAE development(FIG. 34). Thus, the administration of ITE using nanoparticles augmentsits suppressive effect on EAE (compare FIGS. 31 and 33) To study theeffect of ITE-loaded nanoparticles on the T_(reg) compartment we inducedEAE on naïve C57BL/6 mice and treated them, starting at day 0, weeklywith 45 femtomoles of nanoparticles. Spleens were prepared 21 days afterEAE induction and CD4⁺FoxP3⁺ T_(reg) were quantified by FACS.Administration of ITE-loaded nanoparticles led to a significant increasein the number of CD4⁺FoxP3⁺ T_(reg) (FIG. 34); this increase resultedfrom the expansion of both CD25⁺ and CD25⁻ CD4⁺FoxP3⁺T_(reg) (FIG. 34).Thus ITE-loaded nanoparticles can be used to activate AHR and expand theT_(reg) compartment.

To study the mechanism by which the ITE-loaded nanoparticles control EAEwe studied the activity of myelin specific T cells. We induced EAE onnaïve C57BL/6 mice and treated them, starting at day 0, weekly with 45femtomoles of nanoparticles. Spleens were prepared 21 days after EAE wasinduced and analyzed for their recall response to MOG₃₅₋₅₅ and anti-CD3.Mice treated with ITE-loaded nanoparticles showed a suppressed recallproliferative response to the MOG₃₅₋₅₅ peptide (FIG. 35); no differenceswere seen upon activation with antibodies to CD3 (FIG. 35). Whencompared to the splenocytes from control animals, CD4⁺ T cells from micetreated with ITE-loaded nanoparticles secreted higher amounts of TGFb1and IL-10 and lower amounts of IL2, IL6, IFNg and IL17 upon activationwith MOG₃₅₋₅₅ (FIG. 35).

Example 13: Induction of Functional Human Regulatory T Cells by AHRActivation

To investigate the potential of AHR targeting for the induction of humanT_(reg) we activated purified naïve CD4⁺CD62L⁺CD45RO⁻ T cells forhealthy donors for 5 days with antibodies to CD3 and CD28 in thepresence of TCDD 100 nM or TGFb1 2.5 ng/ml or both. T cell activation inthe presence of TCDD resulted in the induction of CD4⁺ FoxP3⁺ T cells insome (FIG. 36) but not all human samples (FIG. 37).

To study the functionality of the putative human T_(reg) induced in thepresence of TCDD we studied their suppressive activity ofCD4⁺CD25^(High) and CD4⁺CD25^(Low) T cells following 5 days ofactivation in the of TCDD or TGFb1 2.5 ng/ml or both. Activation in thepresence of TGFb1 did not result in the induction of suppressive T cells(FIG. 38). However, activation in the presence of TCDD led to thegeneration of both CD4⁺CD25^(High) and CD4⁺CD25^(L)Ow functional humanregulatory T cells, as shown by their ability to inhibit theproliferation of responder T cells (FIG. 38). This effect of TCDD wasamplified in the presence of TGFb1, as shown by the increasedsuppressive activity of the T cells generated under these conditions(FIG. 38).

To investigate the mechanism mediating the suppressive activity of theTCDD-induced T_(reg), we analyzed them by real time PCR for theexpression of several genes that have been previously linked to thesuppressive function of T_(reg). FoxP3 expression was significantlyup-regulated upon activation in the presence of TCDD and TGFb1 (FIG.39), however it was also induced buy TGFb1 alone, suggesting that FoxP3expression does not correlate with the induction of suppressive functionvia AHR activation. This is confirmed by the marginal induction of FoxP3expression triggered by AHR activation with TCDD in the absence of TGFb1(FIG. 39), although these cells expressed low levels of FoxP3 weresuppressive in co-culture assays (FIG. 38). TGFb1 also up-regulated AHRexpression levels several fold over the basal levels observed on Tcells, however the AHR expression levels also did not correlate with theinduction of suppressive activity as shown in co-culture assays (FIG.40). Strikingly, TCDD treated expressed increased levels of IL-10, whichwhere complete inhibited by TGFb1 (FIG. 41). Accordingly, IL-10-specificblocking antibodies could interfere with the suppressive activity ofT_(reg) induced with TCDD, but not the of those induced with TGFb1 andTCDD (FIG. 42). Thus, TCDD-induced CD4⁺CD25^(High) T cells are FoxP3⁻regulatory cells whose suppressive activity is mediated, at leastpartially, via IL-10, resembling the phenotype of type 1 T_(reg)(Roncarolo et al., Immunol Rev. 212, 28 (2006); Roncarolo and Gregori,Eur J Immunol. 38, 925 (2008)).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A composition comprising a ligand that bindsspecifically to an aryl hydrocarbon receptor (AHR) transcription factor,linked to a biocompatible nanoparticle.
 2. The composition of claim 1,wherein the ligand is a small molecule that competes for binding to theAHR competitively with 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) andactivates AHR-dependent signaling.
 3. The composition of claim 1,wherein the ligand is 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD).
 4. Thecomposition of claim 1, wherein the ligand is tryptamine (TA).
 5. Thecomposition of claim 1, further comprising a monoamine oxidase inhibitorsuch as tranylcypromine.
 6. The composition of claim 1, wherein theligand is 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methylester (ITE).
 7. The composition of claim 1, wherein the ligand is6-formylindolo[3,2-b]carbazole (FICZ).
 8. The composition of claim 1,further comprising an antibody that selectively binds to an antigenpresent on a T cell, a B cell, a dendritic cell, or a macrophage.
 9. Thecomposition of claim 8, wherein the antibody is linked to thebiocompatible nanoparticle.
 10. A method for increasing the number ofCD4/CD25/Foxp3-expressing T regulatory (Treg) cells in a population of Tcells, the method comprising: contacting the population of cells with asufficient amount of a composition comprising one or more AHR ligandsselected from the group consisting of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), tryptamine (TA), and2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE), wherein the ligand is linked to a biocompatible nanoparticle, andoptionally evaluating the presence and/or number ofCD4/CD25/Foxp3-expressing cells in the population; wherein the methodresults in an increase in the number and/or activity of regulatory Tcells (Treg).
 11. The method of claim 10, wherein the population of Tcells comprises naïve T cells or CD4⁺CD62 ligand⁺ T cells.
 12. Themethod of claim 10, further comprising administering the Treg cells to asubject suffering from an autoimmune disorder, in an amount sufficientto improve or ameliorate a symptom of the disorder.
 13. The method ofclaim 10, wherein the population of T cells is in a living mammaliansubject.
 14. The method of claim 13, wherein the subject has anautoimmune disorder.
 15. The method of claim 14, wherein the autoimmunedisorder is multiple sclerosis.
 16. The method of claim 13, comprisingadministering the one or more ligands orally.
 17. The method of claim13, comprising administering the one or more ligands intravenously. 18.A method of identifying a candidate compound that increases generationor activity of regulatory T cells (Treg), the method comprising:providing a cell expressing a reporter construct comprising a bindingsequence for the Aryl Hyrocarbon Receptor (AHR) in a mammalian Foxp3promoter sequence, wherein said binding sequence is operably linked to areporter gene, for example a reporter gene selected from the groupconsisting of luciferase, green fluorescent protein, and variantsthereof, contacting the cell with a test compound; and evaluating aneffect of the test compound on expression of the reporter gene, whereina test compound that increases or decreases expression of the reportergene is a candidate compound that modulates generation of Treg.
 19. Themethod of claim 18, further comprising measuring expression of thereporter construct in the presence of a known AHR ligand selected fromthe group consisting of 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD),tryptamine (TA), and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylicacid methyl ester (ITE), or a compound that binds to the AHRcompetitively therewith; determining whether the candidate compoundcompetes for binding to the AHR with the known compound; and selectingthe candidate compound if it binds the AHR competitively with the knowncompound.